Variants of chymosin with improved milk-clotting properties

ABSTRACT

Variants of chymosin with improved milk-clotting properties are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/121,286, filed Aug. 24, 2016, which is the U.S. national stage ofInternational Application No. PCT/EP2015/054020 filed Feb. 26, 2015, andclaims priority to European Patent Application No. 14176664.2 filed Jul.11, 2014, and European Patent Application No. 14156707.3, filed Feb. 26,2014. International Application No. PCT/EP2015/054020 filed Feb. 26,2015 is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 18, 2017, isnamed 030427-0240_SL.txt and is 21,651 bytes in size.

FIELD OF THE INVENTION

The present invention relates to variants of chymosin with improvedmilk-clotting properties.

BACKGROUND ART

Enzymatic coagulation of milk by milk-clotting enzymes, such as chymosinand pepsin, is one of the most important processes in the manufacture ofcheeses. Enzymatic milk coagulation is a two-phase process: a firstphase where a proteolytic enzyme, chymosin or pepsin, attacks κ-casein,resulting in a metastable state of the casein micelle structure and asecond phase, where the milk subsequently coagulates and forms acoagulum.

Chymosin (EC 3.4.23.4) and pepsin (EC 3.4.23.1), the milk clottingenzymes of the mammalian stomach, are aspartic proteases belonging to abroad class of peptidases.

When produced in the gastric mucosal cells, chymosin and pepsin occur asenzymatically inactive pre-prochymosin and pre-pepsinogen, respectively.When chymosin is excreted, an N-terminal peptide fragment, thepre-fragment (signal peptide) is cleaved off to give prochymosinincluding a pro-fragment. Prochymosin is a substantially inactive formof the enzyme which, however, becomes activated under acidic conditionsto the active chymosin by autocatalytic removal of the pro-fragment.This activation occurs in vivo in the gastric lumen under appropriate pHconditions or in vitro under acidic conditions.

The structural and functional characteristics of bovine, i.e. Bostaurus, pre-prochymosin, prochymosin and chymosin have been studiedextensively. The pre-part of the bovine pre-prochymosin moleculecomprises 16 aa residues and the pro-part of the correspondingprochymosin has a length of 42 aa residues. The active bovine chymosincomprises 323 aa is a mixture of two forms, A and B, both of which areactive.

Chymosin is produced naturally in mammalian species such as bovines,camels, caprines, buffaloes, sheep, pigs, humans, monkeys and rats.

Bovine chymosin has for a number of years been commercially available tothe dairy industry.

WO02/36752A2 (Chr. Hansen) describes recombinant production of camelchymosin.

WO2013/174840A1 (Chr. Hansen) describes mutants/variants of bovine andcamel chymosin.

WO2013/164479A2 (DSM) describes mutants of bovine chymosin.

The references listed immediately below may in the present context beseen as references describing mutants of chymosin:

-   Suzuki et al: Site directed mutagenesis reveals functional    contribution of Thr218, Lys220 and Asp304 in chymosin, Protein    Engineering, vol. 4, January 1990, pages 69-71;-   Suzuki et al: Alteration of catalytic properties of chymosin by    site-directed mutagenesis, Protein Engineering, vol. 2, May 1989,    pages 563-569;-   van den Brink et al: Increased production of chymosin by    glycosylation, Journal of biotechnology, vol. 125, September 2006,    pages 304-310;-   Pitts et al: Expression and characterisation of chymosin pH optima    mutants produced in Tricoderma reesei, Journal of biotechnology,    vol. 28, March 1993, pages 69-83;-   M. G. Williams et al: Mutagenesis, biochemical characterization and    X-ray structural analysis of point mutants of bovine chymosin,    Protein engineering design and selection, vol. 10, September 1997,    pages 991-997;-   Strop et al: Engineering enzyme subsite specificity: preparation,    kinetic characterization, and x-ray analysis at 2.0 ANG resolution    of Val111phe site mutated calf chymosin, Biochemistry, vol. 29,    October 1990, pages 9863-9871;-   Supannee et al: Site-specific mutations of calf chymosin B which    influence milk-clotting activity, Food Chemistry, vol. 62, June    1998, pages 133-139;-   Zhang et al: Functional implications of disulfide bond, Cys45-Cys50,    in recombinant prochymosin, Biochimica et biophysica acta, vol.    1343, December 1997, pages 278-286.

None of the prior art references mentioned above describe directly andunambiquously any of the chymosin mutants/variants as described/claimedbelow herein.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to provide variantsof chymosin with improved milk-clotting properties.

As discussed in working examples herein—the present inventors haveidentified a number of improved camel (see Example 6 herein) andbovine/camel (see Example 7 herein) chymosin variants.

Based on a comparative analysis of the camel and bovine variants—thepresent inventors identified a number of further amino acid positionsthat are herein important in the sense that by making a variant in oneor more of these positions one may get an improved chymosin variant.

As known in the art—different natural wildtype chymosin polypeptidesequences obtained from different mammalian species (such as e.g.bovines, camels, sheep, pigs, or rats) are having a relatively highsequence similarity/identity.

In FIG. 1 herein this is exemplified by an alignment of herein relevantdifferent chymosin sequences.

In view of this relatively close sequence relationship—it is believedthat the 3D structures of different natural wildtype chymosins are alsorelatively similar.

In the present context—a natural obtained wildtype chymosin (such asbovine chymosin or camel chymosin) may herein be an example of a parentpolypeptide—i.e. a parent polypeptide to which an alteration is made toproduce a variant chymosin polypeptide of the present invention.

Without being limited to theory—it is believed that the herein discussedchymosin related amino acid positions are of general importance in anyherein relevant chymosin enzyme of interest (e.g. chymosins of e.g.bovines, camels, sheep, pigs, rats etc)—in the sense that by making avariant in one or more of these positions one may get an improvedchymosin variant in general (e.g. an improved bovine, camel, sheep, pigor rat chymosin variant).

As discussed herein—as a reference sequence for determining the aminoacid position of a parent chymosin polypeptide of interest (e.g. camel,sheep, bovine etc) is herein used the public known bovine chymosin Bpreprochymosin sequence (Genbank accession number P00794—disclosed asSEQ ID NO: 1 herein).

The bovine chymosin B preprochymosin of SEQ ID NO: 1 may hereinalternatively be termed Bovine (Bos bovis) chymosin B or simply bovinechymosin. The sequence is also shown in FIG. 1 herein.

Another herein relevant chymosin sequence is publically known Cameliusdrome darius chymosin sequence of SEQ ID NO: 2 herein. It may hereinalternatively be termed camel chymosin. The sequence is also shown inFIG. 1 herein.

In the present context it is believed that a parent chymosin polypeptide(e.g. from sheep or rat) that has at least 65% sequence identity withthe mature polypeptide of SEQ ID NO: 1 (bovine chymosin) may herein beseen as sufficient structural related to e.g. bovine or camel chymosinin order to be improved by making a variant in any of the amino acidpositions as described herein. Accordingly, a first aspect of theinvention relates to a method for making an isolated chymosinpolypeptide variant comprising the steps:

(a): making an alteration at one or more positions in a parentpolypeptide having chymosin activity, wherein the alteration iscomprising a substitution, a deletion or an insertion in at least oneamino acid position corresponding to any of positions 70; 75; 77; 79;90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156, 163; 212; 222;223; 224; 238; 246; 256; 261; K279V; L280; F281;R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309;R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336;346; 361; 367 and 379; and

(b): producing and isolating the altered polypeptide of step (a) andthereby obtaining the isolated chymosin polypeptide variant, wherein thevariant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined byan alignment of the parent polypeptide with the polypeptide of SEQ IDNO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used todetermine the corresponding amino acid sequence in the parentpolypeptide; and

(ii): the parent polypeptide has at least 65% sequence identity with themature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is fromamino acid position 59 to amino acid position 381 of SEQ ID NO: 1;

and with the proviso that the isolated chymosin polypeptide variant isNOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;

R125Q+G128N+H204R+Q246E+S284T;

Y185F+R213Q+Q246E;

V261A+V263I+G309W+L311I+Y326F;

G128D+L188I+Y326F;

G128N+R312S+S313Y+Y326F;

G128N+R312S+S313Y+Y326F;

D117N+V261A+R312S;

D216S+L224V+V263I+F281V+G309D;

Y79S+L224V+L311I; and

R119S+L224V+T297S.

The proviso above may be seen as relating to above discussedWO2013/174840A1 (Chr. Hansen)—since in this document is explicitlydescribed these specific variants.

As understood by the skilled person in the present context—the provisoonly relates to the specific mentioned variants.

For instance, a variant only comprising the Q246E substitution (i.e. notG309D, S329P and/or D337E) is not such a specific variant within theproviso—i.e. it is not disclaimed in the present context.

As known in the art—the skilled person may, based on his common generalknowledge, routinely produce and purify chymosin and chymosin variants.

Said in other words, once the skilled person is in possession of aherein relevant parent polypeptide having chymosin activity of interest(e.g. from bovines, camels, sheep, pigs, or rats) it is routine work forthe skilled person to make a variant of such a parent chymosin ofinterest.

A second aspect of the invention relates to an isolated chymosinpolypeptide variant obtained by the method of first aspect or any hereinrelevant embodiments thereof.

The term “obtained” in relation to the second aspect above should beunderstood as that the isolated chymosin polypeptide variant has beenobtained by the method of first aspect or any herein relevantembodiments thereof.

Accordingly, the term “obtained” in relation to the second aspect shouldnot be understood as obtainable.

As discussed herein—in working examples herein were made variants usingthe polypeptide of SEQ ID NO: 1 (Bovine) as parent polypeptide—suchvariant may herein be termed bovine chymosin variants.

Accordingly, a third aspect of the invention relates to an isolatedchymosin polypeptide variant comprising:

(a): an alteration at one or more positions in a parent polypeptidehaving chymosin activity, wherein the alteration is comprising asubstitution, a deletion or an insertion in at least one amino acidposition corresponding to any of positions 70; 75; 77; 79; 90; 102; 103;108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238;246; 256; 261; K279V; L280; F281;R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309;R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336;346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined byan alignment of the parent polypeptide with the polypeptide of SEQ IDNO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used todetermine the corresponding amino acid sequence in the parentpolypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with themature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is fromamino acid position 59 to amino acid position 381 of SEQ ID NO: 1; and

(iii): the isolated variant polypeptide has less than 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin);

and with the proviso that the isolated chymosin polypeptide variant isNOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;

R125Q+G128N+H204R+Q246E+S284T;

Y185F+R213Q+Q246E;

V261A+V263I+G309W+L311I+Y326F;

G128D+L188I+Y326F;

G128N+R312S+S313Y+Y326F;

G128N+R312S+S313Y+Y326F; and

D117N+V261A+R312S;

D216S+L224V+V263I+F281V+G309D

Y79S+L224V+L311I and

R119S+L224V+T297S.

As discussed herein—in working examples herein were made variants usingthe polypeptide of SEQ ID NO: 2 (camel chymosin) as parentpolypeptide—such variant may herein be termed camel chymosin variant.

Accordingly, a fourth aspect of the invention relates to an isolatedchymosin polypeptide variant comprising:

(a): an alteration at one or more positions in a parent polypeptidehaving chymosin activity, wherein the alteration is comprising asubstitution, a deletion or an insertion in at least one amino acidposition corresponding to any of positions 70; 75; 77; 79; 90; 102; 103;108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238;246; 256; 261; K279V; L280; F281;R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309;R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336;346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined byan alignment of the parent polypeptide with the polypeptide of SEQ IDNO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used todetermine the corresponding amino acid sequence in the parentpolypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with themature polypeptide of SEQ ID NO: 2 (Camel chymosin), which is from aminoacid position 59 to amino acid position 381 of SEQ ID NO: 2; and

(iii): the isolated variant polypeptide has less than 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin);

and with the proviso that the isolated chymosin polypeptide variant isNOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;

R125Q+G128N+H204R+Q246E+S284T;

Y185F+R213Q+Q246E;

V261A+V263I+G309W+L311I+Y326F;

G128D+L188I+Y326F;

G128N+R312S+S313Y+Y326F;

G128N+R312S+S313Y+Y326F;

D117N+V261A+R312S;

D216S+L224V+V263I+F281V+G309D;

Y79S+L224V+L311I; and

R119S+L224V+T297S.

An isolated chymosin polypeptide variant as described herein may be usedaccording to the art—e.g. to make a food or feed product of interest(such as e.g. a milk based product of interest that e.g. could be acheese product).

Accordingly, a fifth aspect of the invention relates to a method formaking a food or feed product comprising adding an effective amount ofthe isolated chymosin polypeptide variant as described herein to thefood or feed ingredient(s) and carrying our further manufacturing stepsto obtain the food or feed product.

Embodiment of the present invention is described below, by way ofexamples only.

Definitions

All definitions of herein relevant terms are in accordance of what wouldbe understood by the skilled person in relation to the herein relevanttechnical context.

The term “chymosin” relates to an enzyme of the EC 3.4.23.4 class.Chymosin has a high specificity and it clots milk by cleavage of asingle 105-Ser-Phe-|-MetAla-108 bond in kappa-chain of casein. Analternative name used in the art is rennin.

The term “chymosin activity” relates to chymosin activity of a chymosinenzyme as understood by the skilled person in the present context.

The skilled person knows how to determine herein relevant chymosinactivity.

In working Example 4 herein is provided an example of a standard methodto determine specific chymosin activity—alternatively termed clottingactivity or milk clotting activity.

In working Example 5 herein is provided an example of a standard methodto determine proteolytical activity.

As known in the art—the herein relevant so-called C/P ratio isdetermined by dividing the specific clotting activity (C) with theproteolytical activity (P).

As known in the art—a higher C/P ratio implies generally that the lossof protein during e.g. cheese manufacturing due to non-specific proteindegradation is reduced, i.e. the yield of cheese is improved, and thatthe development of bitter taste in the cheese during maturation isreduced.

The term “isolated variant” means a variant that is modified by the handof man. In one aspect, the variant is at least 1% pure, e.g., at least5% pure, at least 10% pure, at least 20% pure, at least 40% pure, atleast 60% pure, at least 80% pure, and at least 90% pure, as determinedby SDS PAGE.

The term “mature polypeptide” means a peptide in its final formfollowing translation and any post-translational modifications, such asN terminal processing, C terminal truncation, glycosylation,phosphorylation, etc. In the present context may a herein relevantmature chymosin polypeptide be seen as the active chymosin polypeptidesequence—i.e. without the pre-part and/or pro-part sequences. Hereinrelevant examples of a mature polypeptide are e.g. the maturepolypeptide of SEQ ID NO: 1 (bovine chymosin), which is from amino acidposition 59 to amino acid position 381 of SEQ ID NO: 1 or the maturepolypeptide of SEQ ID NO: 2 (camel chymosin), which is from amino acidposition 59 to amino acid position 381 of SEQ ID NO: 2.

The term “parent” or “parent polypeptide having chymosin activity” meansa polypeptide to which an alteration is made to produce the enzymevariants of the present invention. The parent may be a naturallyoccurring (wild-type) polypeptide or a variant thereof.

The term “Sequence Identity” relates to the relatedness between twoamino acid sequences or between two nucleotide sequences.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the-nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the-nobrief option)is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment).

The term “variant” means a peptide having chymosin activity comprisingan alteration, i.e., a substitution, insertion, and/or deletion, at oneor more (several) positions. A substitution means a replacement of anamino acid occupying a position with a different amino acid; a deletionmeans removal of an amino acid occupying a position; and an insertionmeans adding 1-3 amino acids adjacent to an amino acid occupying aposition.

The amino acid may be natural or unnatural amino acids—for instance,substitution with e.g. a particularly D-isomers (or D-forms) of e.g.D-alanine could theoretically be possible.

The term “wild-type” chymosin peptide means a chymosin expressed by anaturally occurring organism, such as a mammalian (e.g. camel or bovine)found in nature.

DRAWINGS

FIG. 1 : An alignment of herein relevant different chymosin sequences.The shown “Bos_bovis_chymosin_B” is bovine chymosin of SEQ ID NO: 1herein and the shown “Camelus_dromedarius” is camel chymosin of SEQ IDNO: 2 herein. Using bovine chymosin of SEQ ID NO: 1 as referencesequence as described herein is can e.g. be seen that bovine chymosinhas “V” in position 10 and camel chymosin has “A” in the same position10. It may e.g. also be seen that bovine/Rat have “Q” in position 352and Camel/C._bactrianus have “E” in the same position 352. FIG. 1discloses SEQ ID NOS 1, 3, 4, 2, 5 and 6, respectively, in order ofappearance.

In relation to the chymosin sequences shown in FIG. 1 —sheep has 94.5%sequence identity with bovine SEQ ID NO: 1; C._bactrianus has 83.2%sequence identity with bovine SEQ ID NO: 1; Camelus_dromedarius (camelchymosin of SEQ ID NO: 2) has 84% sequence identity with bovine SEQ IDNO: 1; pig has 80.3% sequence identity with bovine SEQ ID NO: 1 and rathas 71.9% sequence with bovine identity SEQ ID NO: 1.

As understood by the skilled person in the present context—hereinrelevant sequence identity percentages of mature polypeptide sequencesof e.g. sheep, C._bactrianus, camel, pig or rat chymosin with the maturepolypeptide of SEQ ID NO: 1 (bovine chymosin—i.e. amino acid positions59 to 381 of SEQ ID NO: 1) are relatively similar to above mentionedsequence identity percentages.

FIG. 2 : The 3D structure of bovine chymosin—the 3D structure is publicavailable. As an example are shown where the amino acid positions 296and 294 are present in bovine Chymosin.

FIG. 3 : Show a graphical representation the REMCAT and Proteol valuesof a number of chymosin variants.

FIG. 4 : PCA plot of effect of individual substitutions. All positionnumbers are 15 lower than numbers used in text.

DETAILED DESCRIPTION OF THE INVENTION

Determining the Amino Acid Position of a Chymosin of Interest

As discussed above—as a reference sequence for determining the aminoacid position of a herein relevant chymosin polypeptide of interest(e.g. camel, sheep, bovine etc.) is herein used the public known bovinechymosin sequence disclosed as SEQ ID NO: 1 herein.

For purposes of the present invention, the polypeptide disclosed in SEQID NO: 1 (bovine chymosin) is used to determine the corresponding aminoacid residue in another chymosin polypeptide. The amino acid sequence ofanother chymosin polypeptide is aligned with the polypeptide disclosedin SEQ ID NO: 1, and based on the alignment, the amino acid positionnumber corresponding to any amino acid residue in the polypeptidedisclosed in SEQ ID NO: 1 is determined using the ClustalW algorithm asdescribed in working Example 1 herein.

Identification of the corresponding amino acid residue in anotherchymosin polypeptide can be confirmed by using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 3.0.0 or later.

Based on above well known computer programs—it is routine work for theskilled person to determine the amino acid position of a herein relevantchymosin polypeptide of interest (e.g. camel, sheep, bovine etc.).

In FIG. 1 herein is shown an example of an alignment.

Just as an example—in FIG. 1 can e.g. be seen that herein used bovinereference SEQ ID NO: 1 has a “G” in position 50 and“Camelus_dromedarius” (SEQ ID NO: 2 herein) has an “A” in this position50.

Nomenclature of Variants

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviations are employed.

The specific variants discussed in this “nomenclature” section below maynot be herein relevant variants of the present invention—i.e. this“nomenclature” section is just to describe the herein relevant usednomenclature as such.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, a theoretical substitution of threonine with alanineat position 226 is designated as “Thr226Ala” or “T226A”. Multiplemutations are separated by addition marks (“+”), e.g.,“Gly205Arg+Ser411Phe” or “G205R+S411F”, representing substitutions atpositions 205 and 411 of glycine (G) with arginine (R) and serine (S)with phenylalanine (F), respectively. A substitution e.g. designated“226A” refers to a substitution of a parent amino acid (e.g. T, Q, S oranother parent amino acid) with alanine at position 226.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G-K-A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of tyrosine and glutamic acidfor arginine and glycine at positions 170 and 195, respectively.

Different substitutions. Where different substitutions can be introducedat a position, the different substitutions are separated by a comma,e.g., “Arg170Tyr,Glu” or “R170Y,E” represents a substitution of argininewith tyrosine or glutamic acid at position 170. Thus,“Tyr167Gly,Ala+Arg170Gly,Ala” or “Y167G,A+R170G,A” designates thefollowing variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

A Method for Making an Isolated Chymosin Polypeptide Variant

As discussed above—as known in the art, the skilled person may, based onhis common general knowledge, routinely produce and purify chymosin andchymosin variants.

Said in other words, once the skilled person is in possession of aherein relevant parent polypeptide having chymosin activity of interest(e.g. from bovines, camels, sheep, pigs, or rats) it is routine work forthe skilled person to make a variant of such a parent chymosin ofinterest.

An example of a suitable method to produce and isolate a chymosin(variant or parent) may be by well known e.g. fungal recombinantexpression/production based technology as e.g. described in WO02/36752A2(Chr. Hansen).

It is also routine work for the skilled person to make alteration at oneor more positions in a parent polypeptide having chymosin activity,wherein the alteration is comprising a substitution, a deletion or aninsertion in at least one amino acid position.

As known to the skilled person—this may e.g. be done by so-called sitedirected mutagenesis and recombinant expression/production basedtechnology.

It is also routine work for the skilled person to determine if a hereinrelevant parent polypeptide (e.g. camel or bovine wildtype chymosin)and/or a herein relevant variant has chymosin activity or not.

As known in the art—chymosin activity may be determined by the so-calledC/P ratio, which is determined by dividing the specific clottingactivity (C) with the proteolytical activity (P).

As known in the art—a higher C/P ratio implies generally that the lossof protein during e.g. cheese manufacturing due to non-specific proteindegradation is reduced, i.e. the yield of cheese is improved, and thatthe development of bitter taste in the cheese during maturation isreduced.

In working example 4 herein is described a suitable method to determinethe specific clotting activity (C) and in working example 5 herein isdescribed a suitable method to determine proteolytical activity (P).

Preferably, an isolated chymosin polypeptide variant as described hereinis a variant, wherein the variant has a chymosin activity giving ahigher C/P ratio as compared to the C/P ratio of bovine chymosincomprising the mature polypeptide of SEQ ID NO: 1 herein.

Preferably, an isolated chymosin polypeptide variant as described hereinis a variant, wherein the variant has a chymosin activity giving ahigher C/P ratio as compared to the C/P ratio of camel chymosincomprising the mature polypeptide of SEQ ID NO: 2 herein.

More preferably, an isolated chymosin polypeptide variant as describedherein is a variant, wherein the variant has

-   -   a chymosin activity giving a higher C/P ratio as compared to the        C/P ratio of bovine chymosin comprising the mature polypeptide        of SEQ ID NO: 1 herein; and    -   a chymosin activity giving a higher C/P ratio as compared to the        C/P ratio of camel chymosin comprising the mature polypeptide of        SEQ ID NO: 2 herein.

As discussed above—as a reference sequence for determining the aminoacid position of a herein relevant chymosin polypeptide of interest(e.g. camel, sheep, bovine etc) is herein used the public known bovinechymosin sequence disclosed as SEQ ID NO: 1 herein.

As discussed above—based on e.g. the computer sequence alignmentprograms discussed herein—it is routine work for the skilled person todetermine the herein relevant amino acid position of a herein relevantchymosin polypeptide of interest (e.g. camel, sheep, bovine etc).

The term “the parent polypeptide has at least 65% sequence identity withthe mature polypeptide of SEQ ID NO: 1 (bovine chymosin)” of e.g. themethod of the first aspect herein may be seen as relating to a sequencebased limitation of the parent chymosin polypeptide used to make aherein relevant variant thereof.

Said in other words—a mature parent chymosin polypeptide (e.g. sheep orpig) that has at least 65% sequence identity with the mature Bovinechymosin is believed to be sufficient structural identical to e.g.Bovine or Camel chymosin in order to be herein relevant—i.e. in thepresent context it is believed that a mature parent chymosin polypeptide(e.g. from e.g. sheep or rat) that has at least 65% sequence identitywith the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) may hereinbe seen as sufficient structural related to e.g. bovine or camelchymosin in order to be improved by making a variant in any of the aminoacid positions as described herein.

The camel chymosin polypeptide of SEQ ID NO: 2 has 84% sequence identitywith the bovine polypeptide of SEQ ID NO: 1 (i.e. the complete SEQ IDNO: 1 from position 1 to 381, which includes pre and pro sequence).

As understood by the skilled person in the present context—a hereinrelevant parent polypeptide having chymosin activity may already e.g. bea variant of e.g. a corresponding wildtype chymosin.

For instance, a camel chymosin variant with e.g. 5-10 alterations (e.g.substitutions) as compared to wildtype camel chymosin polypeptide of SEQID NO: 2 will still be a parent polypeptide that has at least 65%sequence identity with the mature polypeptide of SEQ ID NO: 1 (Bovine)as required in e.g. first aspect herein. Said in other words, a hereinrelevant isolated chymosin polypeptide variant may comprise alterations(e.g. substitutions) in other position than the positions of e.g. thefirst aspect herein.

In relation to the chymosin sequences shown in FIG. 1 herein—sheep has94.5% sequence identity with bovine SEQ ID NO: 1; C._bactrianus has83.2% sequence identity with bovine SEQ ID NO: 1; pig has 80.3% sequenceidentity with bovine SEQ ID NO: 1 and rat has 71.9% sequence with bovineidentity SEQ ID NO: 1.

As understood by the skilled person in the present context—hereinrelevant sequence identity percentages of e.g. mature sheep,C._bactrianus, camel, pig or rat chymosin with the mature polypeptide ofSEQ ID NO: 1 (bovine chymosin—i.e. amino acid positions 59 to 381 of SEQID NO: 1) are relatively similar to above mentioned sequence identitypercentages.

Preferred Variants:

As discussed above—e.g. the first aspect relates to an isolated chymosinpolypeptide variant, wherein the alteration is comprising asubstitution, a deletion or an insertion in at least one amino acidposition corresponding to any of positions 70; 75; 77; 79; 90; 102; 103;108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 223; 224; 238; 246;256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W;G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326;331; 336; 346; 361; 367 and 379.

A preferred embodiment relates to an isolated chymosin polypeptidevariant, wherein the alteration comprises a substitution, a deletion oran insertion in at least one amino acid position corresponding to any ofpositions of e.g. the first aspect herein.

It may be preferred that at least one alteration is a substitution—i.e.a herein relevant preferred embodiment relates to an isolated chymosinpolypeptide variant, wherein the alteration is comprising a substitutionin at least one amino acid position corresponding to any of positionse.g. the first aspect herein.

Preferably, an isolated chymosin polypeptide variant, wherein thealteration is comprising a substitution in at least one amino acidposition corresponding to any of positions L70M; F75Y; K77T; Y79S; V90L;D102N; I103V; K120Q; F124Y; H134Q; I154L; D156V; L163E; S212A; S222G;M223E; L224V; L238I; Q246E; V256I; V261A; K279V; L280I; F281A;R300D,E,S,T,N,Q; R312D,E,S,T,N,Q; E320T; R324V; D325Q; Y326F;K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; 1361L; V367I; orK379P.

Preferably, the substitution is wherein the substitution is Q246E;K279V; R300Q; R312S; Y326F or K336D,E,S,T,N,Q, wherein a preferred K336substitution is K336Q.

As understood by the skilled person in the present context—if the parentchymosin polypeptide already has e.g. “V” in position 156 then is doesnot make sense to talk about making the substitution 156V for thisspecific parent chymosin polypeptide. As can be seen in FIG. 1herein—rat wildtype chymosin has “V” in position 156—the substitution156V may be seen as herein irrelevant for the specific rat chymosinpolypeptide sequence of FIG. 1 .

As understood by the skilled person in the present context—if the parentchymosin polypeptide does not have e.g. “D” in position 156 then is doesnot make sense to talk about making the substitution D156V for thisspecific parent chymosin polypeptide. As can be seen in FIG. 1herein—rat wildtype chymosin has “V” in position 156—the substitutionD156V may therefore be seen as herein irrelevant for the specific ratchymosin polypeptide sequence of FIG. 1 .

In a preferred embodiment, the substitution is wherein the substitutionis:

H134Q+Q246E+Y326F;

D117N+L280I+G309D;

H134Q+D156V+G309D;

D156V+Q246E+L280I;

D117N+H134Q+L280I;

D156V+G309D+Y326F;

D117N+D156V+D325M;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

D117N+H134Q+D325M;

N310Q+N349Q+K279V;

R300Q+N307D;

N307D+G309D;

N307D+R312S;

R300Q+K336Q;

N307D+K336Q;

G309D+R312S;

R300Q+N307D+G309D+R312S+K336Q;

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;

L280I+G309D+S331Y+T342S+D325Q;

L280I+G309D+L224V+E320T+T235S;

L280I+G309W+K77T+R324I;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+Q220S+L224V+H134Q;

L280I+G309W+L238I+T342S;

L280I+G309W+F75Y+Y79S;

L280I+G309D+F75Y+S331Y+Q346E;

L280I+G309D+L224V+I103V+L238I;

L280I+G309D+F124Y+Q346E+I154L;

L280I+G309D+I154L+V261A+V367I;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309D+Y79S+T342S+I154L;

L280I+G309D+Y79S+I103V+F281A;

L280I+G309D+V256I+V261A+K379P;

L280I+G309D+Q346E+K77T+T235S;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y326F+L70M+D325Q;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+S212A+V261A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+K120Q+M223E+H239N;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L; or

L280I+G309W+S212A+V261A.

In a more preferred embodiment, the substitution is wherein thesubstitution is:

D117N+L280I+G309D;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

R300Q+N307D+G309D+R312S+K336Q; or

N158Q+N349Q+R300Q+N307D+G309D+R312MS=K336Q.

Preferred Parent Polypeptide Having Chymosin Activity:

Preferably, the parent polypeptide has at least 70% sequence identitywith the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), morepreferably the parent polypeptide has at least 75% sequence identitywith the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

Just as an example—a herein suitable relevant parent polypeptide coulde.g. be bovine chymosin A—as known in the art bovine chymosin A may onlyhave one amino acid difference as compared to bovine chymosin B of SEQID NO: 1 herein.

As discussed above—in working examples herein were made variants usingthe polypeptide of SEQ ID NO: 1 (Bovine) as parent polypeptide—suchvariant may herein be termed bovine chymosin variants.

Accordingly, in a preferred embodiment—the parent polypeptide has atleast 90% sequence identity with the mature polypeptide of SEQ ID NO: 1(bovine chymosin), more preferably the parent polypeptide has at least95% sequence identity with the mature polypeptide of SEQ ID NO: 1(bovine chymosin) and even more preferably the parent polypeptide has atleast 97% sequence identity with the mature polypeptide of SEQ ID NO: 1(bovine chymosin). It may be preferred that the parent polypeptide isthe mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

As understood by the skilled person in the present context—a hereinrelevant parent polypeptide having chymosin activity may already e.g. bea variant of e.g. a corresponding wildtype chymosin.

For instance, a bovine chymosin variant with e.g. 5-10 alterations (e.g.substitutions) as compared to mature wildtype bovine chymosinpolypeptide of SEQ ID NO: 1 will still be a parent polypeptide that hasat least 95% sequence identity with the mature polypeptide of SEQ ID NO:1 (Bovine chymosin).

The mature polypeptide of SEQ ID NO: 1 (Bovine) is 323 amino acidslong—accordingly, a bovine chymosin variant with e.g. 25 amino acidsubstitutions as compared to mature wildtype bovine chymosin polypeptideof SEQ ID NO: 1 will not be a parent polypeptide that has at least 95%sequence identity with the mature polypeptide of SEQ ID NO: 1 (Bovinechymosin).

Said in other words and in general—a herein relevant isolated chymosinpolypeptide variant may comprise alterations (e.g. substitutions) inother positions than the positions of e.g. the first aspect herein.

As discussed above—in working examples herein were made variants usingthe polypeptide of SEQ ID NO: 2 (Camel) as parent polypeptide—suchvariant may herein be termed camel chymosin variant.

Accordingly, in a preferred embodiment—the parent polypeptide has atleast 90% sequence identity with the mature polypeptide of SEQ ID NO: 2(Camel chymosin), more preferably the parent polypeptide has at least95% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camelchymosin) and even more preferably the parent polypeptide has at least97% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camelchymosin). It may be preferred that the parent polypeptide is the maturepolypeptide of SEQ ID NO: 2 (Camel chymosin).

As understood by the skilled person in the present context—a parentpolypeptide that has at least 90% sequence identity with the maturepolypeptide of SEQ ID NO: 2 (Camel) is still within the SEQ ID NO: 1(Bovine) based sequence identity requirement of point (ii) of firstaspect herein—i.e. it will be a parent polypeptide that has at least 65%sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovinechymosin).

An Isolated Variant of Bovine Chymosin:

As discussed above—in working examples herein were made variants usingthe polypeptide of SEQ ID NO: 1 (Bovine) as parent polypeptide—suchvariant may herein be termed bovine chymosin variants.

As discussed above—the third aspect accordingly relates to an isolatedchymosin polypeptide variant comprising:

(a): an alteration at one or more positions in a parent polypeptidehaving chymosin activity, wherein the alteration is comprising asubstitution, a deletion or an insertion in at least one amino acidposition corresponding to any of positions 70; 75; 77; 79; 90; 102; 103;108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238;246; 256; 261; K279V; L280; F281;R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309;R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336;346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined byan alignment of the parent polypeptide with the polypeptide of SEQ IDNO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used todetermine the corresponding amino acid sequence in the parentpolypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with themature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is fromamino acid position 59 to amino acid position 381 of SEQ ID NO: 1; and

(iii): the isolated variant polypeptide has less than 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin);

and with the proviso that the isolated chymosin polypeptide variant isNOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;

R125Q+G128N+H204R+Q246E+S284T;

Y185F+R213Q+Q246E;

V261A+V263I+G309W+L311I+Y326F;

G128D+L188I+Y326F;

G128N+R312S+S313Y+Y326F;

G128N+R312S+S313Y+Y326F; and

D117N+V261A+R312S;

D216S+L224V+V263I+F281V+G309D

Y79S+L224V+L311I and

R119S+L224V+T297S.

The above described definitions and preferred embodiments are alsorelevant for this aspect.

Preferably, an isolated bovine chymosin polypeptide variant as describedherein is a variant, wherein the variant has a chymosin activity givinga higher C/P ratio as compared to the C/P ratio of bovine chymosincomprising the mature polypeptide of SEQ ID NO: 1.

In a preferred embodiment—the parent polypeptide has at least 92%sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovinechymosin), more preferably the parent polypeptide has at least 95%sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovinechymosin) and even more preferably the parent polypeptide has at least97% sequence identity with the mature polypeptide of SEQ ID NO: 1(bovine chymosin). It may be preferred that the parent polypeptide isthe mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

As understood by the skilled person in the present context—an isolatedchymosin variant may comprise alterations (e.g. substitutions) in otheramino acid positions than given above.

For instance, a bovine chymosin variant with e.g. 5-10 alterations (e.g.substitutions) as compared to wildtype bovine chymosin polypeptide ofSEQ ID NO: 1 will still be a parent polypeptide that has at least 95%sequence identity with the mature polypeptide of SEQ ID NO: 1 (Bovinechymosin).

It may be preferred that the isolated bovine chymosin variant comprisesless than 30 amino acid alterations (e.g. substitutions) as compared tothe mature polypeptide of SEQ ID NO: 1 (bovine chymosin) or it may bepreferred that the isolated bovine chymosin variant comprises less than20 amino acid alterations (e.g. substitutions) as compared to the maturepolypeptide of SEQ ID NO: 1 (bovine chymosin) or it may be preferredthat the isolated bovine chymosin variant comprises less than 10 aminoacid alterations (e.g. substitutions) as compared to the maturepolypeptide of SEQ ID NO: 1 (bovine chymosin) or it may be preferredthat the isolated bovine chymosin variant comprises less than 5 aminoacid alterations (e.g. substitutions) as compared to the maturepolypeptide of SEQ ID NO: 1 (bovine chymosin).

As understood by the skilled person in the present context—the term “theisolated variant polypeptide has less than 100% sequence identity withthe mature polypeptide of SEQ ID NO: 1 (bovine chymosin)” of point (iii)above relates to that the herein described isolated bovine chymosinvariant shall of course not have a polypeptide sequence that is 100%identical to the public known wildtype bovine chymosin sequence of SEQID NO: 1.

A preferred embodiment relates to an isolated bovine chymosinpolypeptide variant, wherein the alteration comprises a substitution, adeletion or an insertion in at least one amino acid positioncorresponding to any of positions of the third aspect.

It may be preferred that at least one alteration is a substitution—i.e.a herein relevant preferred embodiment relates to an isolated chymosinpolypeptide variant, wherein the alteration is comprising a substitutionin at least one amino acid position corresponding to any of positions ofthe third aspect.

Preferably, the substitution is L70M; F75Y; K77T; Y79S; V90L; D102N;I103V; N108D; D117N; F114Y; K120Q; F124Y; H134Q; I154L; D156V; L163E;S212A; M223E; L224V; L238I; Q246E; V256I; V261A; K279V; L280I; F281A;R300D,E,S,T,N,Q; R312D,E,S,T,N,Q; E320T; R324V; D325Q; Y326F;K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; 1361L; V367I; orK379P.

Preferably, the substitution is Q246E; K279V; R300Q; R312S; Y326F orK336D,E,S,T,N,Q, wherein a preferred K336 substitution is K336Q.

In a preferred embodiment, the substitution is:

H134Q+Q246E+Y326F;

D117N+L280I+G309D;

H134Q+D156V+G309D;

D156V+Q246E+L280I;

D117N+H134Q+L280I;

D156V+G309D+Y326F;

D117N+D156V+D325M;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

D117N+H134Q+D325M;

N310Q+N349Q+K279V;

R300Q+N307D;

N307D+G309D;

N307D+R312S;

R300Q+K336Q;

N307D+K336Q;

G309D+R312S;

R300Q+N307D+G309D+R312S+K336Q;

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;

L280I+G309D+S331Y+T342S+D325Q;

L280I+G309D+L224V+E320T+T235S;

L280I+G309W+K77T+R324I;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+Q220S+L224V+H134Q;

L280I+G309W+L238I+T342S;

L280I+G309W+F75Y+Y79S;

L280I+G309D+F75Y+S331Y+Q346E;

L280I+G309D+L224V+I103V+L238I;

L280I+G309D+F124Y+Q346E+I154L;

L280I+G309D+I154L+V261A+V367I;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309D+Y79S+T342S+I154L;

L280I+G309D+Y79S+I103V+F281A;

L280I+G309D+V256I+V261A+K379P;

L280I+G309D+Q346E+K77T+T235S;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y326F+L70M+D325Q;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+S212A+V261A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+K120Q+M223E+H239N;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L; or

L280I+G309W+S212A+V261A.

In a more preferred embodiment, the substitution is wherein thesubstitution is:

D117N+L280I+G309D;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

R300Q+N307D+G309D+R312S+K336Q; or

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.

An Isolated Variant of Camel Chymosin:

As discussed above—in working examples herein were made variants usingthe polypeptide of SEQ ID NO: 2 (camel chymosin) as parentpolypeptide—such variant may herein be termed camel chymosin variant.

As discussed above—the fourth aspect accordingly relates to an isolatedchymosin polypeptide variant comprising:

(a): an alteration at one or more positions in a parent polypeptidehaving chymosin activity, wherein the alteration is comprising asubstitution, a deletion or an insertion in at least one amino acidposition corresponding to any of positions 70; 75; 77; 79; 90; 102; 103;108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 223; 224; 238; 246;256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W;G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326;331; 336; 346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined byan alignment of the parent polypeptide with the polypeptide of SEQ IDNO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used todetermine the corresponding amino acid sequence in the parentpolypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with themature polypeptide of SEQ ID NO: 2 (Camel chymosin), which is from aminoacid position 59 to amino acid position 381 of SEQ ID NO: 2; and

(iii): the isolated variant polypeptide has less than 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin);

and with the proviso that the isolated chymosin polypeptide variant isNOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;

R125Q+G128N+H204R+Q246E+S284T;

Y185F+R213Q+Q246E;

V261A+V263I+G309W+L311I+Y326F;

G128D+L188I+Y326F;

G128N+R312S+S313Y+Y326F;

G128N+R312S+S313Y+Y326F;

D117N+V261A+R312S;

D216S+L224V+V263I+F281V+G309D;

Y79S+L224V+L311I; and

R119S+L224V+T297S.

The above described definitions and preferred embodiments are alsorelevant for this aspect.

Preferably, an isolated camel chymosin polypeptide variant as describedherein is a variant, wherein the variant has a chymosin activity givinga higher C/P ratio as compared to the C/P ratio of camel chymosincomprising the mature polypeptide of SEQ ID NO: 2.

In a preferred embodiment—the parent polypeptide has at least 92%sequence identity with the mature polypeptide of SEQ ID NO: 2 (camelchymosin), more preferably the parent polypeptide has at least 95%sequence identity with the mature polypeptide of SEQ ID NO: 2 (camelchymosin) and even more preferably the parent polypeptide has at least97% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camelchymosin). It may be preferred that the parent polypeptide is the maturepolypeptide of SEQ ID NO: 2 (Camel chymosin).

As understood by the skilled person in the present context—an isolatedchymosin variant may comprise alterations (e.g. substitutions) in otheramino acid positions than given above.

For instance, a camel chymosin variant with e.g. 5-10 alterations (e.g.substitutions) as compared to wildtype camel chymosin polypeptide of SEQID NO: 2 will still be a parent polypeptide that has at least 95%sequence identity with the mature polypeptide of SEQ ID NO: 2 (camelchymosin).

It may be preferred that the isolated camel chymosin variant comprisesless than 30 amino acid alterations (e.g. substitutions) as compared tothe mature polypeptide of SEQ ID NO: 2 (camel chymosin) or it may bepreferred that the isolated camel chymosin variant comprises less than20 amino acid alterations (e.g. substitutions) as compared to the maturepolypeptide of SEQ ID NO: 2 (camel chymosin) or it may be preferred thatthe isolated camel chymosin variant comprises less than 10 amino acidalterations (e.g. substitutions) as compared to the mature polypeptideof SEQ ID NO: 2 (camel chymosin) or it may be preferred that theisolated camel chymosin variant comprises less than 5 amino acidalterations (e.g. substitutions) as compared to the mature polypeptideof SEQ ID NO: 2 (camel chymosin).

As understood by the skilled person in the present context—the term “theisolated variant polypeptide has less than 100% sequence identity withthe mature polypeptide of SEQ ID NO: 2 (camel chymosin)” of point (iii)above relates to that the herein described isolated camel chymosinvariant shall of course not have a polypeptide sequence that is 100%identical to the public known wildtype camel chymosin sequence of SEQ IDNO: 2.

A preferred embodiment relates to an isolated camel chymosin polypeptidevariant, wherein the alteration comprises a substitution, a deletion oran insertion in at least one amino acid position corresponding to any ofpositions of the fourth aspect.

It may be preferred that at least one alteration is a substitution—i.e.a herein relevant preferred embodiment relates to an isolated chymosinpolypeptide variant, wherein the alteration is comprising a substitutionin at least one amino acid position corresponding to any of positions ofthe fourth aspect.

Preferably, the substitution is L70M; F75Y; K77T; Y79S; V90L; D102N;I103V; K120Q; F124Y; I154L; L163E; S212A; M223E; L224V; L238I; Q246E;V256I; V261A; K279V; R300D,E,S,T,N,Q; R312D,E,S,T,N,Q; E320T; R324V;Y326F; K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; 1361L;V367I; or K379P.

Preferably, the substitution is Q246E; K279V; R300Q; R312S; Y326F orK336D,E,S,T,N,Q, wherein a preferred K336 substitution is K336Q.

In a preferred embodiment, the substitution is:

H134Q+Q246E+Y326F;

D117N+L280I+G309D;

H134Q+D156V+G309D;

D156V+Q246E+L280I;

D117N+H134Q+L280I;

D156V+G309D+Y326F;

D117N+D156V+D325M;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

D117N+H134Q+D325M;

N310Q+N349Q+K279V;

R300Q+N307D;

N307D+G309D;

N307D+R312S;

R300Q+K336Q;

N307D+K336Q;

G309D+R312S;

R300Q+N307D+G309D+R312S+K336Q;

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;

L280I+G309D+S331Y+T342S+D325Q;

L280I+G309D+L224V+E320T+T235S;

L280I+G309W+K77T+R324I;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+Q220S+L224V+H134Q;

L280I+G309W+L238I+T342S;

L280I+G309W+F75Y+Y79S;

L280I+G309D+F75Y+S331Y+Q346E;

L280I+G309D+L224V+I103V+L238I;

L280I+G309D+F124Y+Q346E+I154L;

L280I+G309D+I154L+V261A+V367I;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309D+Y79S+T342S+I154L;

L280I+G309D+Y79S+I103V+F281A;

L280I+G309D+V256I+V261A+K379P;

L280I+G309D+Q346E+K77T+T235S;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y326F+L70M+D325Q;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+S212A+V261A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+K120Q+M223E+H239N;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L; or

L280I+G309W+S212A+V261A.

In a more preferred embodiment, the substitution is wherein thesubstitution is:

D117N+L280I+G309D;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

R300Q+N307D+G309D+R312S+K336Q; or

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.

A Method for Making a Milk Based Product

As discussed above—an isolated chymosin polypeptide variant as describedherein may be used according to the art—e.g. to make a milk basedproduct of interest (such as e.g. a cheese product).

As discussed above—an aspect of the invention relates to a method formaking a food or feed product comprising adding an effective amount ofthe isolated chymosin polypeptide variant as described herein to thefood or feed ingredient(s) and carrying our further manufacturing stepsto obtain the food or feed product.

Preferably, the food or feed product is a milk based product and whereinthe method comprises adding an effective amount of the isolated chymosinpolypeptide variant as described herein to milk and carrying our furthermanufacturing steps to obtain the milk based product.

The milk may e.g. be soy milk, sheep milk, goat milk, buffalo milk, yakmilk, lama milk, camel milk or cow milk.

The milk based product may e.g. be a fermented milk product, a quark ora cheese.

Aspects/Embodiments Herein—Presented in Claim Format

Herein described aspects and preferred embodiments of the invention maybe presented/described in a so-called claim format—this is done below.

1. A method for making an isolated chymosin polypeptide variantcomprising the steps:

(a): making an alteration at one or more positions in a parentpolypeptide having chymosin activity, wherein the alteration iscomprising a substitution, a deletion or an insertion in at least oneamino acid position corresponding to any of positions 70; 75; 77; 79;90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222;223; 224; 238; 246; 256; 261; K279V; L280; F281;R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309;R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336;346; 361; 367 and 379; and

(b): producing and isolating the altered polypeptide of step (a) andthereby obtaining the isolated chymosin polypeptide variant, wherein thevariant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined byan alignment of the parent polypeptide with the polypeptide of SEQ IDNO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used todetermine the corresponding amino acid sequence in the parentpolypeptide; and

(ii): the parent polypeptide has at least 65% sequence identity with themature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is fromamino acid position 59 to amino acid position 381 of SEQ ID NO: 1;

and with the proviso that the isolated chymosin polypeptide variant isNOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;

R125Q+G128N+H204R+Q246E+S284T;

Y185F+R213Q+Q246E;

V261A+V263I+G309W+L311I+Y326F;

G128D+L188I+Y326F;

G128N+R312S+S313Y+Y326F;

G128N+R312S+S313Y+Y326F;

D117N+V261A+R312S;

D216S+L224V+V263I+F281V+G309D;

Y79S+L224V+L311I; and

R119S+L224V+T297S.

2. The method for making an isolated chymosin polypeptide variant ofclaim 1, wherein the isolated chymosin polypeptide variant has:

-   -   a chymosin activity giving a higher C/P ratio as compared to the        C/P ratio of bovine chymosin comprising the mature polypeptide        of SEQ ID NO: 1; and    -   a chymosin activity giving a higher C/P ratio as compared to the        C/P ratio of camel chymosin comprising the mature polypeptide of        SEQ ID NO: 2.

3. The method for making an isolated chymosin polypeptide variant of anyof the preceding claims, wherein the alteration comprises asubstitution, a deletion or an insertion in at least one amino acidposition corresponding to any of positions of claim 1.

4. The method for making an isolated chymosin polypeptide variant of anyof the preceding claims, wherein the alteration comprises a substitutionin at least one amino acid position corresponding to any of positions ofclaim 1.

5. The method for making an isolated chymosin polypeptide variant ofclaim 4, wherein the substitution is L70M; F75Y; K77T; Y79S; V90L;D102N; I103V; N108D; D117N; F114Y; K120Q; F124Y; H134Q; I154L; D156V;L163E; S212A; S222G; M223E; L224V; L238I; Q246E; V256I; V261A; K279V;L280I; F281A; R300D,E,S,T,N,Q; R312D,E,S,T,N,Q; E320T; R324V; D325Q;Y326F; K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; 1361L;V367I; or K379P.

6. The method for making an isolated chymosin polypeptide variant ofclaim 5, wherein the substitution is Q246E; K279V; R300Q; R312S; Y326For K336D,E,S,T,N,Q.

7. The method for making an isolated chymosin polypeptide variant ofclaim 6, wherein the substitution is K336Q.

8. The method for making an isolated chymosin polypeptide variant ofclaim 4, wherein the substitution is:

H134Q+Q246E+Y326F;

D117N+L280I+G309D;

H134Q+D156V+G309D;

D156V+Q246E+L280I;

D117N+H134Q+L280I;

D156V+G309D+Y326F;

D117N+D156V+D325M;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

D117N+H134Q+D325M;

N310Q+N349Q+K279V;

R300Q+N307D;

N307D+G309D;

N307D+R312S;

R300Q+K336Q;

N307D+K336Q;

G309D+R312S;

R300Q+N307D+G309D+R312S+K336Q;

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;

L280I+G309D+S331Y+T342S+D325Q;

L280I+G309D+L224V+E320T+T235S;

L280I+G309W+K77T+R324I;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+Q220S+L224V+H134Q;

L280I+G309W+L238I+T342S;

L280I+G309W+F75Y+Y79S;

L280I+G309D+F75Y+S331Y+Q346E;

L280I+G309D+L224V+I103V+L238I;

L280I+G309D+F124Y+Q346E+I154L;

L280I+G309D+I154L+V261A+V367I;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309D+Y79S+T342S+I154L;

L280I+G309D+Y79S+I103V+F281A;

L280I+G309D+V256I+V261A+K379P;

L280I+G309D+Q346E+K77T+T235S;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y326F+L70M+D325Q;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+S212A+V261A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+K120Q+M223E+H239N;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L; or

L280I+G309W+S212A+V261A.

9. The method for making an isolated chymosin polypeptide variant ofclaim 4, wherein the substitution is:

D117N+L280I+G309D;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

R300Q+N307D+G309D+R312S+K336Q; or

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.

10. The method for making an isolated chymosin polypeptide variant ofany of the preceding claims, wherein the parent polypeptide has at least75% sequence identity with the mature polypeptide of SEQ ID NO: 1(bovine chymosin).

11. The method for making an isolated chymosin polypeptide variant ofclaim 10, wherein the parent polypeptide has at least 95% sequenceidentity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

12. The method for making an isolated chymosin polypeptide variant ofany of claims 1 to 9, wherein the parent polypeptide has at least 95%sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camelchymosin), which is from amino acid position 59 to amino acid position381 of SEQ ID NO: 2.

13. An isolated chymosin polypeptide variant obtained by the method ofany of claims 1 to 12.

14. An isolated chymosin polypeptide variant comprising:

(a): an alteration at one or more positions in a parent polypeptidehaving chymosin activity, wherein the alteration is comprising asubstitution, a deletion or an insertion in at least one amino acidposition corresponding to any of positions 70; 75; 77; 79; 90; 102; 103;108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238;246; 256; 261; K279V; L280; F281;R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309;R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336;346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined byan alignment of the parent polypeptide with the polypeptide of SEQ IDNO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used todetermine the corresponding amino acid sequence in the parentpolypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with themature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is fromamino acid position 59 to amino acid position 381 of SEQ ID NO: 1; and

(iii): the isolated variant polypeptide has less than 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin);

and with the proviso that the isolated chymosin polypeptide variant isNOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;

R125Q+G128N+H204R+Q246E+S284T;

Y185F+R213Q+Q246E;

V261A+V263I+G309W+L311I+Y326F;

G128D+L188I+Y326F;

G128N+R312S+S313Y+Y326F;

G128N+R312S+S313Y+Y326F;

D117N+V261A+R312S;

D216S+L224V+V263I+F281V+G309D

Y79S+L224V+L311I and

R119S+L224V+T297S.

15. The isolated chymosin polypeptide variant of claim 14, wherein theisolated variant has a chymosin activity giving a higher C/P ratio ascompared to the C/P ratio of bovine chymosin comprising the maturepolypeptide of SEQ ID NO: 1.

16. The isolated chymosin polypeptide variant of any of claims 14 to 15,wherein the parent polypeptide has at least 97% sequence identity withthe mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

17. The isolated chymosin polypeptide variant of any of claims 14 to 16,wherein the isolated bovine chymosin variant comprises less than 10amino acid alterations (e.g. substitutions) as compared to the maturepolypeptide of SEQ ID NO: 1 (bovine chymosin).

18. The isolated chymosin polypeptide variant of any of claims 14 to 17,wherein the alteration comprises a substitution, a deletion or aninsertion in at least one amino acid position corresponding to any ofpositions of claim 14.

19. The isolated chymosin polypeptide variant of any of claims 16 to 18,wherein the alteration comprises a substitution in at least one aminoacid position corresponding to any of positions of claim 14.

20. The isolated chymosin polypeptide variant of claim 19, wherein thesubstitution is L70M; F75Y; K77T; Y79S; V90L; D102N; I103V; N108D;D117N; F114Y; K120Q; F124Y; H134Q; I154L; D156V; L163E; S212A; S222G;M223E; L224V; L238I; Q246E; V256I; V261A; K279V; L280I; F281A;R300D,E,S,T,N,Q; G309D,W; R312D,E,S,T,N,Q; E320T; R324V; D325Q; Y326F;K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; I361L; V367I; orK379P.

21. The isolated chymosin polypeptide variant of claim 20, wherein thesubstitution is Q246E; K279V; R300Q; R312S; Y326F or K336D,E,S,T,N,Q.

22. The isolated chymosin polypeptide variant of claim 21, wherein thesubstitution is K336Q.

23. The isolated chymosin polypeptide variant of claim 19, wherein thesubstitution is:

H134Q+Q246E+Y326F;

D117N+L280I+G309D;

H134Q+D156V+G309D;

D156V+Q246E+L280I;

D117N+H134Q+L280I;

D156V+G309D+Y326F;

D117N+D156V+D325M;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

D117N+H134Q+D325M;

N310Q+N349Q+K279V;

R300Q+N307D;

N307D+G309D;

N307D+R312S;

R300Q+K336Q;

N307D+K336Q;

G309D+R312S;

R300Q+N307D+G309D+R312S+K336Q;

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;

L280I+G309D+S331Y+T342S+D325Q;

L280I+G309D+L224V+E320T+T235S;

L280I+G309W+K77T+R324I;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+Q220S+L224V+H134Q;

L280I+G309W+L238I+T342S; L280I+G309W+F75Y+Y79S;

L280I+G309D+F75Y+S331Y+Q346E;

L280I+G309D+L224V+I103V+L238I;

L280I+G309D+F124Y+Q346E+I154L;

L280I+G309D+I154L+V261A+V367I;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309D+Y79S+T342S+I154L;

L280I+G309D+Y79S+I103V+F281A;

L280I+G309D+V256I+V261A+K379P;

L280I+G309D+Q346E+K77T+T235S;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y326F+L70M+D325Q;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+S212A+V261A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+K120Q+M223E+H239N;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L; or

L280I+G309W+S212A+V261A.

24. The isolated chymosin polypeptide variant of claim 19, wherein thesubstitution is:

D117N+L280I+G309D;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

R300Q+N307D+G309D+R312S+K336Q; or

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.

25. An isolated chymosin polypeptide variant comprising:

(a): an alteration at one or more positions in a parent polypeptidehaving chymosin activity, wherein the alteration is comprising asubstitution, a deletion or an insertion in at least one amino acidposition corresponding to any of positions 70; 75; 77; 79; 90; 102; 103;108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238;246; 256; 261; K279V; L280; F281;R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309;R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336;346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined byan alignment of the parent polypeptide with the polypeptide of SEQ IDNO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used todetermine the corresponding amino acid sequence in the parentpolypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with themature polypeptide of SEQ ID NO: 2 (Camel chymosin), which is from aminoacid position 59 to amino acid position 381 of SEQ ID NO: 2; and

(iii): the isolated variant polypeptide has less than 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin);

and with the proviso that the isolated chymosin polypeptide variant isNOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;

R125Q+G128N+H204R+Q246E+S284T;

Y185F+R213Q+Q246E;

V261A+V263I+G309W+L311I+Y326F;

G128D+L188I+Y326F;

G128N+R312S+S313Y+Y326F;

G128N+R312S+S313Y+Y326F;

D117N+V261A+R312S;

D216S+L224V+V263I+F281V+G309D;

Y79S+L224V+L311I; and

R119S+L224V+T297S.

26. The isolated chymosin polypeptide variant of claim 25, wherein theisolated variant has a chymosin activity giving a higher C/P ratio ascompared to the C/P ratio of camel chymosin comprising the maturepolypeptide of SEQ ID NO: 2.

27. The isolated chymosin polypeptide variant of any of claims 25 to 26,wherein the parent polypeptide has at least 97% sequence identity withthe mature polypeptide of SEQ ID NO: 2 (camel chymosin).

28. The isolated chymosin polypeptide variant of any of claims 25 to 26,wherein the isolated camel chymosin variant comprises less than 10 aminoacid alterations (e.g. substitutions) as compared to the maturepolypeptide of SEQ ID NO: 2 (camel chymosin).

29. The isolated chymosin polypeptide variant of any of claims 25 to 28,wherein the alteration comprises a substitution, a deletion or aninsertion in at least one amino acid position corresponding to any ofpositions of claim 23.

30. The isolated chymosin polypeptide variant of any of claims 25 to 29,wherein the alteration comprises a substitution in at least one aminoacid position corresponding to any of positions of positions of claim23.

31. The isolated chymosin polypeptide variant of claim 30, wherein thesubstitution is L70M; F75Y; K77T; Y79S; V90L; D102N; I103V; N108D;D117N; F114Y; K120Q; F124Y; H134Q; I154L; D156V, L163E; S212A; S222G;M223E; L224V; D325Q; L238I; Q246E; V256I; V261A; K279V; L280I; F281A;R300D,E,S,T,N,Q; G309D,W; R312D,E,S,T,N,Q; E320T; R324V; Y326F;K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; 1361L; V367I; orK379P.

32. The isolated chymosin polypeptide variant of claim 31, wherein thesubstitution is Q246E; K279V; R300Q; R312S; Y326F or K336D,E,S,T,N,Q.

33. The isolated chymosin polypeptide variant of claim 32, wherein thesubstitution is K336Q.

34. The isolated chymosin polypeptide variant of claim 30, wherein thesubstitution is:

H134Q+Q246E+Y326F;

D117N+L280I+G309D;

H134Q+D156V+G309D;

D156V+Q246E+L280I;

D117N+H134Q+L280I;

D156V+G309D+Y326F;

D117N+D156V+D325M;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

D117N+H134Q+D325M;

N310Q+N349Q+K279V;

R300Q+N307D;

N307D+G309D;

N307D+R312S;

R300Q+K336Q;

N307D+K336Q;

G309D+R312S;

R300Q+N307D+G309D+R312S+K336Q;

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;

L280I+G309D+S331Y+T342S+D325Q;

L280I+G309D+L224V+E320T+T235S;

L280I+G309W+K77T+R324I;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+Q220S+L224V+H134Q;

L280I+G309W+L238I+T342S;

L280I+G309W+F75Y+Y79S;

L280I+G309D+F75Y+S331Y+Q346E;

L280I+G309D+L224V+I103V+L238I;

L280I+G309D+F124Y+Q346E+I154L;

L280I+G309D+I154L+V261A+V367I;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309D+Y79S+T342S+I154L;

L280I+G309D+Y79S+I103V+F281A;

L280I+G309D+V256I+V261A+K379P;

L280I+G309D+Q346E+K77T+T235S;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y326F+L70M+D325Q;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+S212A+V261A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+K120Q+M223E+H239N;

L280I+G309D+H239N+R324I+D325Q;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L;

L280I+G309D+L70M+T342S;

L280I+G309D+H134Q+V213F+F281A;

L280I+G309D+Y79S+L224V+S212A;

L280I+G309D+S331Y+L224V+Y326F;

L280I+G309D+H134Q+M223E+L70M;

L280I+G309W+L238I+T342S;

L280I+G309D+V213F+E320T+V90L; or

L280I+G309W+S212A+V261A.

35. The isolated chymosin polypeptide variant of claim 30, wherein thesubstitution is:

D117N+L280I+G309D;

L280I+D325M+Y326F;

D117N+Q246E+Y326F;

R300Q+N307D+G309D+R312S+K336Q; or

N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.

36: A method for making a food or feed product comprising adding aneffective amount of the isolated chymosin polypeptide variant accordingto any of claims 14 to 35 to the food or feed ingredient(s) and carryingour further manufacturing steps to obtain the food or feed product.

37: The method for making a food or feed product of claim 35, whereinthe product is a milk based product and wherein the method comprisesadding an effective amount of the isolated chymosin polypeptide variantaccording to any of claims 14 to 36 to milk and carrying our furthermanufacturing steps to obtain the milk based product.

38: The method for making a milk based product of claim 37, wherein themilk is soy milk, sheep milk, goat milk, buffalo milk, yak milk, lamamilk, camel milk or cow milk.

39: The method for making a milk based product of any of claims 36 to38, wherein the milk based product is a fermented milk product, a quarkor a cheese.

EXAMPLES Example 1: Alignment and Numbering of Chymosin ProteinSequences and Variant Sequences

Chymosin protein sequences were aligned using the ClustalW algorithm asprovided by the EBI (EBI, tools, multiple sequence alignment, CLUSTALW”,http://www.ebi.ac.uk/Tools/msa/clustalw2/) and as described in Larkin MA, Blackshields G, Brown N P, Chenna R, McGettigan P A, McWilliam H,Valentin F, Wallace I M, Wilm A, Lopez R, Thompson J D, Gibson T J,Higgins D G (2007). Bioinformatics 23(21), 2947-2948.

ClustalW2 settings for multiple sequence alignments were Protein weightMatrix=BLOSUM, GAP open=10, GAP EXTENSION=0,05, GAP DISTANCES=8, No EndGaps, ITERATION=none, NUMITER=1, CLUSTERING=NJ

As a reference sequence the bovine chymosin B preprochymosin was used(Genbank accession number P00794—disclosed herein as SEQ ID NO: 1),where the N-terminal Methionin has number 1 (MRCL . . . ) (SEQ ID NO: 7)and the C-terminal Isoleucin (in the protein sequence . . . LAKAI) (SEQID NO: 8) has number 381. Variants were aligned against the bovine Bpre-pro-chymosin and residues were numbered according to thecorresponding bovine chymosin residue.

Example 2: Design of Chymosin Variants

Chymosin variants were designed using different strategies.

When there is referred to camel chymosin there is referred to camelchymosin comprising the polypeptide of SEQ ID NO: 2 herein.

Camel chymosin of SEQ ID NO: 2 may be seen as a herein relevant parentpolypeptide having chymosin activity used to make camel chymosinvariants thereof.

When there is referred to bovine chymosin there is referred to bovinechymosin comprising the polypeptide of SEQ ID NO: 1 herein.

Bovine chymosin of SEQ ID NO: 1 may be seen as a herein relevant parentpolypeptide having chymosin activity used to make bovine chymosinvariants thereof. Variants of camel chymosin were designed based on analignment of a large set of public known aspartic protease sequenceshaving an identity of 25% or more compared to bovine chymosin B.

Variations were generally introduced in hypervariable regions, whileconserved regions were not changed. Multiple variations were introducedin each variant construct, ensuring that each single mutation waspresent in multiple variant constructs (for discussion of results—seeexample 6 below).

Variants of bovine chymosin were designed based on a comparison ofbovine and camel chymosin. Bovine residues were e.g. changed to thecamel counterpart (for discussion of results—see example 7 below).

Example 3: Preparation of Chymosin Variant Enzyme Material

All chymosin variants were synthesized as synthetic genes and clonedinto a fungal expression vector corresponding essentially to pGAMpR-C(described in WO02/36752A2)

The vectors were transformed into E. coli and plasmid DNA was purifiedusing standard molecular biology protocols, known to the person skilledin the art. The variant plasmids were individually transformed into anAspergillus niger or Aspergillus nidulans strain and protein wasproduced essentially as described in WO02/36752A2 and purified usingstandard chromatography techniques.

As known in the art—the skilled person may, based on his common generalknowledge, produce and purify chymosin and chymosin variants—such asherein described bovine and camel chymosin variants.

Example 4: Determination of Specific Chvmosin Activity

4.1 Determination of Clotting Activity

Milk clotting activity was determined using the REMCAT method, which isthe standard method developed by the International Dairy Federation (IDFmethod) Milk clotting activity is determined from the time needed for avisible flocculation of a standard milk substrate prepared from alow-heat, low fat milk powder with a calcium chloride solution of 0.5 gper liter (pH≈6.5). The clotting time of a rennet sample is compared tothat of a reference standard having known milk-clotting activity andhaving the same enzyme composition by IDF Standard 110B as the sample.Samples and reference standards were measured under identical chemicaland physical conditions. Variant samples were adjusted to approximately3 IMCU/ml using an 84 mM acetic acid pH 5.5 buffer. Hereafter, 200 μlenzyme was added to 10 ml preheated milk (32° C.) in a glass test tubeplaced in a water bath, capable of maintaining a constant temperature of32° C.±1° C. under constant stirring.

The total milk-clotting activity (strength) of a rennet was calculatedin International Milk-Clotting Units (IMCU) per ml relative to astandard having the same enzyme composition as the sample according tothe formula:Strength in IMCU/ml=Sstandard×Tstandard×Dsample/Dstandard×Tsample

-   -   Sstandard: The milk-clotting activity of the international        reference standard for rennet.    -   Tstandard: Clotting time in seconds obtained for the standard        dilution.    -   Dsample: Dilution factor for the sample    -   Dstandard: Dilution factor for the standard    -   Tsample: Clotting time in seconds obtained for the diluted        rennet sample from addition of enzyme to time of flocculation

For clotting activity determination of camel variant evaluation inExample 9, the μIMCU method was used instead of the REMCAT method. Ascompared to REMCAT, flocculation time of chymosin variants in the μIMCUassay was determined by OD measurements in 96-well microtiter plates at800 nm in a UV/VIS plate reader. A standard curve of various dilutionsof a reference standard with known clotting strength was recorded oneach plate. Samples were prepared by diluting enzyme in 84 mM acetatebuffer, 0.1% triton X-100, pH 5.5. Reaction at 32° C. was started byadding 250 uL of a standard milk substrate containing 4% (w/w) low-heat,low fat milk powder and 7.5% (w/w) calcium chloride (pH≈6.5) to 25 uLenzyme sample. Milk clotting activity of chymosin variants inInternational Milk-Clotting Units (IMCU) per ml was determined based onsample flocculation time relative to the standard curve.

4.2 Determination of Total Protein Content

Total protein content was determined using the Pierce BCA Protein AssayKit from Thermo Scientific following the instructions of the providers.

4.3 Calculation of Specific Clotting Activity

Specific clotting activity (IMCU/mg total protein) was determined bydividing the clotting activity (IMCU/ml) by the total protein content(mg total protein per ml).

Example 5: Determination of Proteolytic Activity

General proteolytic activity was measured using fluorescently labelledBodipy-FL casein as a substrate (EnzChek; Molecular Bioprobes, E6638).Casein derivatives heavily labeled with pH-insensitive green-fluorescentBodipy-FL result in almost complete quenching of the conjugate'sfluorescence. Protease catalyzed hydrolysis releases fluorescentBodipy-FL. This method is very sensitive which was essential for thisexperiment as CHYMAX M has the lowest general proteolytical activity ofall coagulants known to date.

The assay was conducted in a 0.2 M phosphate buffer adjusted to thedesired pH at a final substrate concentration of 0.04 mg/ml. Prior tomixing 1 part of substrate with 1 part of enzyme, both prepared in thephosphate buffer, all enzyme variants where normalized to 50 IMCU/ml(according to Example 4). The substrate and enzyme were mixed in a96-well Nunc Fluoro microtitter plates, sealed and incubated at 32° C.for 60 min. After incubation the sealing was removed and thefluorescence recorded in a fluorimeter. For variants evaluated inExamples 9 and 10, 1 part of substrate was mixed with 1 part ofnon-normalized enzyme samples in 386-well Nunc Fluoro microtitter platesand the fluorescence was continuously recorded in a fluorimeter at 32 Cfor 10 hours. Slopes of the linear part of fluorescence increase wereused to determine general proteolytic activity.

Example 6: Evaluation of Camel Chymosin Variants

For all variants the specific clotting activity (IMCU/mg of totalprotein) was determined at pH 6.5 according to Example 4 and theproteolytical activity was determined according to example 5 at pH 6.5The C/P ratio was determined for all variants at pH 6.5 by dividing thespecific clotting activity (IMCU/mg) with the proteolytical activity.

As a reference the camel wildtype gene was included.

Variants with Multiple Substitutions

It can be concluded that there are clear combinatorial effects, wheredifferent substitutions have an effect on the respective effects.

IMCU/mg Proteol C/P 1 H134Q, Q246E, Y326F 104% 211%  49% 2 D117N, L280I,G309D 122%  66% 185% 3 H134Q, D156V, G309D 117% 179%  66% 4 D156V,Q246E, L280I 105% 199%  53% 5 D117N, H134Q, L280I  67% 683%  10% 6D156V, G309D, Y326F 100% 115%  87% 8 D117N, D156V, D325M 127% 457%  28%9 L280I, D325M, Y326F 113%  94% 121% 10 D117N, Q246E, Y326F 127% 121%105% 11 D117N, H134Q, D325M 134% 192%  69% Ref came1 100% 100% 100%

It can be concluded that variants 1, 2, 3, 4, 8, 9, 10 and 11 have ahigher specific milk clotting activity, with variants 2, 8, 10 and 11having the strongest improvement

It can be concluded that variants 2 and 9 have a reduced proteolyticalactivity.

It can be concluded that variants 2, 9 and 10 have an increased C/Pratio.

Based on this variant 2 is the most preferred variant, while variants 9and 10 also show preferred characteristics.

Individual Mutations

As all variants included multiple mutations, the data of the rankedvariants were investigated in more details using statistical methods and3D structure analysis, to determine the individual amino acid changesthat have a positive or negative effect.

The effects of the individual amino acid changes can be summarized asfollows but depend much upon the other amino acid changes in thedifferent variants. Based on these the preferred mutations are D117N,Q246E, G 309D, Y326F and L280I.

C P C/P H134Q + −− −− Exposed lobe Q246E + −− − Backbone Y326F + − +/−Backbone D117N ++ −− − Backbone lobe L280I + +/− +/− In cleft G309D + −−+/− Outside small lobe D156V + −− −− Backbone D325M ++ −− −− BackboneThe term “+” refers to a positive amino acid exchange - i.e. “++” ismore positive than “+”. The term “−” refers to a negative amino acidexchange - i.e.“−−” is more negative than “−” The term “positive” refersto a positive effect on the cheese making properties of the variants,i.e. improved clotting activity (“C”) and increased C/P ratio areconsidered to be positive (“+” or “++”) while increased generalproteolytical activity (“P”) is considered to be a negative property(“−” or “−−”) The qualification “+/−” indicates a relatively neutraleffect

The descriptions of the right column of the table relates to where theindividual mutations are situated in the 3D structure of camel chymosin.The 3D structure of camel chymosin is publicly available.

Conclusions:

The results above demonstrate that following individual mutations incamel chymosin were preferred (i.e. with improved C/P ratio as comparedto camel wildtype chymosin): D117N, Q246E, G 309D, Y326F, L280I.

The results above demonstrate that following multiplesubstitutions/mutations in camel chymosin were preferred (i.e. withimproved C/P ratio as compared to camel wildtype chymosin):

D117N+L280I+G309D;

L280I+D325M+Y326F;

D117N+Q246E+Y326F.

Example 7: Evaluation of Camel and Bovine Chymosin Variants

For all variants the specific clotting activity (IMCU/mg of totalprotein) was determined at pH 6.5 according to Example 4, while thegeneral or aspecific proteolytical activity was determined as describedin example 5.

The C/P ratio was determined for all variants at pH 6.5 by dividing thespecific clotting activity (IMCU/mg) with the proteolytical activity.

As a reference a camel wildtype gene was included.

For better comparison all variants were made in a background that didnot have active N-glycosylation sites, the so called Ugly variants.These were made by changing the N in the two potential N-glycosylationsites into a Q.

For further results, see FIG. 3 .

Description of the Variants

In variant J2, K279 was replaced by V in bovine non-glycosylatedchymosin In variant J32, the flap region from bovine non-glycosylatedchymosin was replaced by the flap region from Pepsin.

In variant J72, the negative patch from bovine chymosin was used toreplace the corresponding regions in camel chymosin. In variant J44,R300 was replaced in camel chymosin by Q, the corresponding amino acidin bovine chymosin. This mutation is also found in variant J72.

Relative to camel RemCat Prot C/P J2 BovUgly N310Q, N349Q, K279V  54%227%  24% J22 BovUgly Pepsin positive patch  15% 115%  13% J32 BovUglyK279V, L80I, K129E,  6%  63%  10% P130T, H134T, Q141T, V171F, E191S,Y192G, N310Q, N349Q J44 CamUGly R300Q 123% 114% 108% J72 CamUgly N158Q,N349Q, R300Q, 125%  58% 215% N307D, G309D, R312S, K336Q CamUgly CamelN158Q, N349Q 100%  98% 102% BovUgly Bovine N310Q, N349Q  40% 208%  19%Camel N/A 100% 100% 100%

Conclusions:

Mutation of the Lysin at position 279 of bovine chymosin resulted in avariant that showed comparable proteolytical activity and an increasedspecific clotting activity as compared to bovine chymosin (variant J2).Accordingly, it can be concluded that Valine at position 279 is thepreferred amino acid.

The effect of glycosylation of Camel chymosin on the cheese makingproperties is neglectible. Comparison of the unglycosylated camelvariant with the wildtype camel chymosin indicates no significantchanges. However, introduction of the negative patch reason from bovinechymosin in camel chymosin (variant J72) shows a positive effect on thespecific clotting activity, while the general proteolytical activity isapproximately 2 fold reduced, resulting in a doubling of the C/P ratio.Introduction of the single mutation R300Q from this patch (variant J44)shows a similar positive effect on clotting activity as seen for variantJ72. Q is concluded to be the preferred amino acid in position 300.

The negative patch region in bovine chymosin is expected to have animportant effect for positioning of the enzyme outward the correctcleavage site, thus improving the enzymes specificity. The effect isexpected to be mostly charge related, i.e. any change that increases thenegative charge in this reason will result in increased specificity.

Below is shown an alignment of the negative charged region of bovine andcamel chymosin. Only charged residues are indicated.

Camel (SEQ ID NO: 9) RxxxxxxNxGxxRxxxxxxxxxxxxxxxxxxxxxxxK Bovine(SEQ ID NO: 10) QxxxxxxDxDxxSxxxxxxxxxxxxxxxxxxxxxxxQ

With respect to position numbers and using the Camel as reference thenumbering is starting from the right:

R300

N307

G309

R312

K336

Example 8: Evaluation of Camel Variants

A number of different variants, each having multiple substitutions ascompared to the wild type camel chymosin, was analyzed.

For all variants the specific clotting activity (IMCU/mg of totalprotein) was determined at pH 6.5 according to Example 4, while theaspecific proteolytical activity was determined as described in Example5 by measuring proteolytical activity per 100 IMCU.

As a reference a camel wildtype gene was included.

Analysis of Variants

The variants indicated in the table have an amino acid sequenceidentical to the camel chymosin gene (indicated by camel wt), except forthe variations mentioned for each variant.

Clotting activity is mentioned as IMCU per mg of total protein. Improvedclotting activities are indicated with one or more “+” symbols.Proteolytical activity is expressed in artificial units per 100 IMCU.Improved variants, i.e. variants with reduced proteolytical activities,are indicated with one or more “+” symbols. More “+” symbols indicate astronger improvement. In the “Overall” column “+” symbols indicatevariants that have generally improved properties, i.e. a lowproteolytical activity with a high clotting activity.

TABLE 1 analysis of camel chymosin variants Clotting ProteolyticalIMCU/mg AU/100 IMCU Overall 1 L280I G309D E141S Q220S R324I 196 161852 2L280I G309W F75Y Y79S 419 ++ 43355 3 L280I G309D H134Q S222G S331Y 29936409 4 L280I G309D K120Q M223E H239N 250 13642 +++ 5 L280I G309D Q220SV213F T342S 231 139775 6 L280I G309D H134Q V213F F281A 376 + 23575 + 7L280I G309D S331Y L224V Y326F 318 12257 ++++ + 8 L280I G309D Y326F V241IE305T 353 + 33477 9 L280I G309D S331Y F124Y I346L 338 37156 10 L280IG309D M223E L224V L273V 324 36425 11 L280I G309D H134Q M223E L70M 386 +10664 ++++ ++ 12 L280I G309D F75Y S331Y Q346E 418 ++ 40393 13 L280IG309D L224V I103V L238I 412 ++ 50010 14 L280I G309W L238I T342S 420 +++21087 + ++ 15 L280I G309D L70M T342S 395 ++ 22743 + 16 L280I G309D Y79SL224V S212A 403 ++ 23684 + 17 L280I G309D V213F E320T V90L 426 +++21956 + ++ 18 L280I G309D L163E S222G V261A 246 97468 19 L280I G309WS212A V261A 344 10865 ++++ + 20 L280I G309D Q220S L224V H134Q 425 +++35156 21 L280I G309W K77T R324I 434 +++ 45616 22 L280I G309W I361L I103V324 32966 23 L280I G309D E141S R324V V367I 360 + 77215 24 L280I G309DY79S L273V L163E 317 62132 25 L280I G309D I154L T235S K379P 333 93587 26L280I G309D F75Y T342S V261A 361 + 108877 27 L280I G309D V90L K379PV318T 317 52280 28 L280I G309D V256I V90L E141S 289 81720 29 L280I G309DI154L V261A V367I 405 ++ 59055 30 L280I G309D Y326F L273V V90L 312 5483331 L280I G309D H134Q L163E V318T 344 43594 32 L280I G309D Y79S H134QY326F 337 30815 33 L280I G309D Y79S I103V F281A 379 + 104307 34 L280IG309D V256I V261A K379P 378 + 39517 35 L280I G309D S331Y L238I I154L 29360312 36 L280I G309D S222G R324V I154L 223 62784 37 L280I G309D H239NF124Y V90L 312 55432 38 L280I G309D H239N R324I D325Q 377 + 17261 ++ 39L280I G309W K120Q V367I 354 75440 40 L280I G309D Y326F L70M D325Q 373 +72792 41 L280I G309D L224V E320T T235S 446 +++ 32453 42 L280I G309DS331Y T342S D325Q 475 ++++ 70103 43 L280I G309D F124Y Q346E I154L 410 ++33586 44 L280I G309D V261A R324V F281A 198 34974 45 L280I G309D I361LS212A V318T 343 64876 46 L280I G309D Y79S T342S I154L 382 + 122413 47L280I G309D Q346E K77T T235S 377 + 34716 48 L280I G309D K120Q Y326F K77T264 46463 Camel wt 366 + 15.664 +++ Bovine wt 208 62.662

High specific clotting activity is essential for a good milk clottingenzymes. In total 21 variants with an increased specific clottingactivity, relative to the camel chymosin, were identified and includedin Table 2 below.

TABLE 2 Camel chymosin variants with increased Clotting activityClotting Proteolytical Overall 42 L280I G309D S331Y T342S D325Q 475 ++++70.103 41 L280I G309D L224V E320T T235S 446 +++ 30.953 21 L280I G309WK77T R324I 434 +++ 45.616 17 L280I G309D V213F E320T V90L 426 +++21.956 + ++ 20 L280I G309D Q220S L224V H134Q 425 +++ 35.156 14 L280IG309W L238I T342S 420 +++ 21.087 + ++ 2 L280I G309W F75Y Y79S 419 ++43.355 12 L280I G309D F75Y S331Y Q346E 418 ++ 40.393 13 L280I G309DL224V I103V L238I 412 ++ 50.010 43 L280I G309D F124Y Q346E I154L 410 ++33.586 29 L280I G309D I154L V261A V367I 405 ++ 59.055 16 L280I G309DY79S L224V S212A 403 ++ 23.684 + 15 L280I G309D L70M T342S 395 ++22.743 + 11 L280I G309D H134Q M223E L70M 386 + 10.664 ++++ ++ 46 L280IG309D Y79S T342S I154L 382 + 122.413 33 L280I G309D Y79S I103V F281A379 + 104.307 34 L280I G309D V256I V261A K379P 378 + 39.517 47 L280IG309D Q346E K77T T235S 377 + 34.716 38 L280I G309D H239N R324I D325Q377 + 17.261 ++ 6 L280I G309D H134Q V213F F281A 376 + 23.575 + 40 L280IG309D Y326F L70M D325Q 373 + 72.792 Camel 366 + 15.664 +++ wt

Reduced proteolytical activity is a perquisite for a good milk clottingenzymes. In total 10 variants with a reduced proteolytical activity,relative to the camel chymosin, were identified (see Table 3 below).

TABLE 3 Camel chymosin variants with reduced proteolytical activityClotting Proteolyical Overall 11 L280I G309D H134Q M223E L70M 386 +10.664 ++++ ++ 19 L280I G309W S212A V261A 344 10.865 ++++ + 7 L280IG309D S331Y L224V Y326F 318 12.257 ++++ + 4 L280I G309D K120Q M223EH239N 250 13.642 +++ 38 L280I G309D H239N R324I D325Q 377 + 17.261 ++ 14L280I G309W L238I T342S 420 +++ 21.087 + ++ 17 L280I G309D V213F E320TV90L 426 +++ 21.956 + 15 L280I G309D L70M T342S 395 ++ 22.743 + 6 L280IG309D H134Q V213F F281A 376 + 23.575 + 16 L280I G309D Y79S L224V S212A403 ++ 23.684 + Camel 366 + 15.664 +++ wt

Based on an overall analysis five variants were identified that hadimproved properties for both milk clotting and proteolytical activities.These five variants are indicated in table 4 below.

TABLE 4 Camel chymosin variants with increased clotting activity anddecreased proteolytical activity Clotting Proteolytical Overall 7 L280IG309D S331Y L224V Y326F 318 12.257 ++++ + 11 L280I G309D H134Q M223EL70M 386 + 10.664 ++++ ++ 14 L280I G309W L238I T342S 420 +++ 21.087 + ++17 L280I G309D V213F E320T V90L 426 +++ 21.956 + ++ 19 L280I G309W S212AV261A 344 10.865 ++++ + Camel 366 + 15.664 +++ wt

Statistical Analysis of the Effects of Individual Mutations

A statistical, PCA based, analysis was used to identify single mutationswith positive effects on either proteolytical activity, milk clottingactivity, or both. In the table below, mutations resulting in increasedclotting activity, decreased proteolytical activity or both increasedclotting and decreased proteolytical activity are summarized. The PCAplot is indicated in the FIG. 4 .

TABLE 5 single substitutions having positive effects on clotting,proteolytical activity or on both Clotting + proteolytical ClottingProteolytical H134Q I103V R324V L224V F75Y K120Q Q346E D325Q M223E L70MI154L S331Y G309W I361L K379P E320T Y79S L163E L238I D117N V90L L280IV367I V261A V256I K77T S212A F124Y

Positional Effects

It was expected that most mutations that would have an effect onclotting activity or on general proteolytical activity (i.e.specificity) would be located in or close to the catalytical cleft. Thesubstrate is entering the catalytical cleft and it is also here thatcleavage takes place.

Surprisingly, only few of the substitutions that were shown to have apositive effect on clotting activity and/or specificity were located inthis region (for example L280I L70M and F75Y). Many mutations that had apositive effect were found on other parts of the molecule

Substitutions Resulting in Improved Clotting Activity

Most of the substitutions resulting in improved clotting activity werelocated in the body of the enzyme and are likely to have causedconformational changes in the molecule. Substitution F75Y is located atthe entrance of the cleft and is rather subtle, resulting in increasedpolarity.

TABLE 6 substitutions giving improved clotting I103V Lobe, back F75YCleft entrance D325Q Backbone I154L Backbone I361L Body Y79S BackboneD117N Side L280I Close to cleft V261A Side

Substitutions Resulting in Reduced Proteolytical Activity

Most of the substitutions are located in the body of the molecule. Theresulting conformational changes might result in increased accessibilityfor the substrate. Two mutations were found at the lobes that mark theentrance of the catalytical cleft. The L163E substitution increases thenegative charge. This strengthens the results from example 7, showingthe importance of charge in these positions.

TABLE 6 Mutations resulting in reduced proteolytical activity R324VBackbone K120Q Side M223E Body S331Y Lobe K379P Backbone L163E Lobe

Substitutions Resulting in Improved Clotting and Reduced ProteolyticalActivity

Some of the substitutions that result in an overall improvement of themilk clotting capabilities result in charge changes that are likely tobe involved in substrate recognition. These include H134Q resulting inhigher positive, as well as the Q346E substitution resulting in morenegative charge. Other substitutions with positive effects on bothclotting and specificity are most likely resulting in more generalconformational changes of the chymosin molecule.

TABLE 7 Mutations giving improved clotting and reduced proteolyticalactivity H134Q Outside flap L224V Backbone Q346E Entrance cleft L70MCleft G309W Side lobe E320T Backbone L238I Backbone V90L Close to cleftV367I Backbone V256I Backbone K77T Side protruding S212A Backbone F124YBackbone

Example 9: Evaluation of Camel Variants

Variant Characterization

Camel chymosin variants evaluated in Example 7 regarding their milkclotting (C) and general proteolytic (P) activities were produced againand evaluated regarding their casein cleavage specificity C/P (Table 1below). The C/P ratio is a measure for a coagulant's efficiency incheese making, i.e., the yield of cheese curd obtained from a certainvolume of milk. Milk clotting and general proteolytic activities weredetermined as described in Examples 4 and 5, respectively. In thisexample, however, proteolytic activity was measured withoutnormalization for clotting activity.

Camel chymosin was analyzed as reference. C/P values of all variants areshown as relative values to wild type camel chymosin. An impact of totalprotein concentration in the enzyme samples on C/P was detected, and C/Pvalues were corrected for this correlation accordingly.

TABLE 1 Analysis of camel chymosin variants variant mutations Clotting(C) Proteolytical (P) C/P 1 L280I G309D E141S Q220S R324I  92% 125%  25%2 L280I G309W F75Y Y79S 108% 129%  78% 3 L280I G309D H134Q S222G S331Y103%  34% 271% 4 L280I G309D K120Q M223E H239N  96%  81%  85% 5 L280IG309D Q220S V213F T342S  75% 113%  42% 6 L280I G309D H134Q V213F F281A 62%  31% 339% 7 L280I G309D S331Y L224V Y326F  91% 110% 143% 8 L280IG309D Y326F V241I E305T 135% 114%  94% 9 L280I G309D S331Y F124Y I346L 98% 123%  81% 10 L280I G309D M223E L224V L273V  93%  78% 105% 11 L280IG309D H134Q M223E L70M 116%  68% 246% 12 L280I G309D F75Y S331Y Q346E155%  83% 172% 13 L280I G309D L224V I103V L238I 136%  89% 128% 14 L280IG309W L238I T342S 124% 159%  89% 15 L280I G309D L70M T342S  93% 152% 35% 16 L280I G309D Y79S L224V S212A 137%  91% 100% 17 L280I G309D V213FE320T V90L 133% 163%  46% 18 L280I G309D L163E S222G V261A  72%  49%182% 19 L280I G309W S212A V261A 104% 122% 138% 20 L280I G309D Q220SL224V H134Q 201%  52% 315% 21 L280I G309W K77T R324I 160% 102% 139% 22L280I G309W I361L I103V 108% 132%  79% 24 L280I G309D Y79S L273V L163E 91%  76% 112% 25 L280I G309D I154L T235S K379P 112% 118% 112% 26 L280IG309D F75Y T342S V261A 108%  90% 141% 27 L280I G309D V90L K379P V318T 95% 135%  55% 28 L280I G309D V256I V90L E141S 109% 146% 139% 29 L280IG309D I154L V261A V367I 157%  95% 156% 30 L280I G309D Y326F L273V V90L 99% 119%  58% 31 L280I G309D H134Q L163E V318T  95%  59% 247% 32 L280IG309D Y79S H134Q Y326F 105%  66% 219% 33 L280I G309D Y79S I103V F281A124%  66% 342% 34 L280I G309D V256I V261A K379P 146% 102% 134% 36 L280IG309D S222G R324V I154L  76%  68% 161% 37 L280I G309D H239N F124Y V90L102% 125%  67% 38 L280I G309D H239N R324I D325Q  90% 143% 127% 39 L280IG309W K120Q V367I 103%  94% 139% 40 L280I G309D Y326F L70M D325Q  96%207%  10% 41 L280I G309D L224V E320T T235S 116% 102% 134% 42 L280I G309DS331Y T342S D325Q 145% 102% 158% 43 L280I G309D F124Y Q346E I154L 135% 94% 176% 44 L280I G309D V261A R324V F281A  71%  63% 137% 45 L280I G309DI361L S212A V318T 116% 122% 100% 46 L280I G309D Y79S T342S I154L 137%102% 115% 47 L280I G309D Q346E K77T T235S 124% 107% 123% 48 L280I G309DK120Q Y326F K77T  90%  86% 113% Camel wt 100% 100% 100%

A total of 30 out of 46 characterized variants show improved C/Pcompared to wild type camel chymosin (Table 2 below). A more than 3-foldimprovement was observed for the three top variants 33, 6 and 20.

TABLE 2 Camel chymosin variants with improved C/P variant mutationsClotting (C) Proteolytical (P) C/P 33 L280I G309D Y79S I103V F281A 124% 66% 342% 6 L280I G309D H134Q V213F F281A  62%  31% 339% 20 L280I G309DQ220S L224V H134Q 201%  52% 315% 3 L280I G309D H134Q S222G S331Y 103% 34% 271% 31 L280I G309D H134Q L163E V318T  95%  59% 247% 11 L280I G309DH134Q M223E L70M 116%  68% 246% 32 L280I G309D Y79S H134Q Y326F 105% 66% 219% 18 L280I G309D L163E S222G V261A  72%  49% 182% 43 L280I G309DF124Y Q346E I154L 135%  94% 176% 12 L280I G309D F75Y S331Y Q346E 155% 83% 172% 36 L280I G309D S222G R324V I154L  76%  68% 161% 42 L280I G309DS331Y T342S D325Q 145% 102% 158% 29 L280I G309D I154L V261A V367I 157% 95% 156% 7 L280I G309D S331Y L224V Y326F  91% 110% 143% 26 L280I G309DF75Y T342S V261A 108%  90% 141% 21 L280I G309W K77T R324I 160% 102% 139%28 L280I G309D V256I V90L E141S 109% 146% 139% 39 L280I G309W K120QV367I 103%  94% 139% 19 L280I G309W S212A V261A 104% 122% 138% 44 L280IG309D V261A R324V F281A  71%  63% 137% 34 L280I G309D V256I V261A K379P146% 102% 134% 41 L280I G309D L224V E320T T235S 116% 102% 134% 13 L280IG309D L224V I103V L238I 136%  89% 128% 38 L280I G309D H239N R324I D325Q 90% 143% 127% 47 L280I G309D Q346E K77T T235S 124% 107% 123% 46 L280IG309D Y79S T342S I154L 137% 102% 115% 48 L280I G309D K120Q Y326F K77T 90%  86% 113% 24 L280I G309D Y79S L273V L163E  91%  76% 112% 25 L280IG309D I154L T235S K379P 112% 118% 112% 10 L280I G309D M223E L224V L273V 93%  78% 105% Camel wt 100% 100% 100%

Statistical Analysis of the Positional and Mutational Effects on C/P

A statistical, PCA based, analysis was used to identify single mutationswith positive effects on the specificity of milk clotting over generalcasein proteolysis (C/P) of camel chymosin. The following mutations werefound to be beneficial for high C/P ratios:

H134Q, F281A, H103V, V256I, I154L, S222G, L224V, Q346E, S331Y, K77T,V367I, G309D, V261A, D325Q, L280, D117N, L163E, S212A

Example 10: Evaluation of Camel Variants

Variant Characterization

Based on the positional and mutational effects determined in Example 7,another set of camel chymosin variants was generated with multiplesubstitutions as compared to wild type camel chymosin and evaluatedregarding their casein substrate specificity (C/P) as described inExample 9 (Table 1 below).

TABLE 1 Analysis of camel chymosin variants variant mutations Clotting(C) Proteolytical (P) C/P 1 L70M Y79S D117N H134Q M223E V256I L280IG309D Q346E 132% 116% 117% 2 L70M Y79S D117N H134Q M223E L280I G309WS331Y 131%  56% 194% 3 L70M D117N H134Q M223E V256I L280I G309D S331YK379P 109%  75% 135% 4 L70M D117N H134Q S212A M223E V261A L280I G309DV367I  83% 115% 108% 5 L70M D117N H134Q D156V L280I 135% 108% 137% 6L70M K77T V90L D117N H134Q D202Q M223E L280I G309D 135% 113% 124% 7 L70MY79S D117N H134Q M223E V261A L280I G309D E320T 141% 124% 143% 8 L70MV109L H134Q M223E G309D  82%  86%  87% 9 L70M D117N F124Y H134Q M223EL238I L280I G309D V367I 105%  97% 115% 10 L70M D117N H134Q S212A M223EL280I G309W Q346E 101%  79% 133% 11 L70M D117N H134Q D156V M223E L280IG309D E320T Q346E 153% 101% 119% 12 L70M V109L D117N H134Q L224V L280IG309D  98%  71% 128% 13 L70M D117N H134Q D202Q M223E V261A L280I 116%144% 126% 14 L70M D117N D202Q M223E L224V L280I G309D  85% 126% 111% 15L70M K77T D117N H134Q S212A M223E V256I L280I G309D 154% 130% 129% 16L70M H134Q D156V M223E L280I G309W 136% 131% 137% 17 L70M V90L D117NH134Q M223E L238I V256I L280I G309D 121% 101%  97% 18 L70M D117N H134QS212A M223E S331Y 124%  76% 151% 19 L70M V109L D117N F124Y H134Q M223EV261A L280I G309W  96%  98% 128% 20 L70M V90L H134Q M223E L280I E320T138% 110%  98% 21 L70M N108D D117N H134Q M223E G309W E320T 187% 151%138% 22 V109L D117N H134Q M223E L238I L280I G309D E320T 110%  93% 106%23 L70M D117N H134Q M223E G309D Q346E V367I K379P  67% 102% 118% 24 L70MN108D D117N V261A L280I G309D  95% 117% 102% 25 L70M D117N H134Q L238IL280I G309W K379P  97%  92% 113% 26 L70M Y79S D117N M223E L280I K379P137% 123% 129% 27 D117N H134Q M223E L224V V256I L280I 132% 102% 127% 28L70M K77T N108D D117N H134Q M223E L280I Q346E 167% 106% 166% 29 L70MY79S N108D D117N F124Y H134Q D202Q M223E L280I G309D 183%  57% 151%Bovine wt 100% 100% 100%

A total of 26 out of 29 variants show improved C/P ratios, as comparedto wild type camel chymosin. A 2-fold improvement was observed for thebest variant (Table 2, below).

TABLE 2 Camel chymosin variants with improved C/P variant mutationsClotting (C) Proteolytical (P) C/P 2 L70M Y79S D117N H134Q M223E L280IG309W S331Y 131%  56% 194% 28 L70M K77T N108D D117N H134Q M223E L280IQ346E 167% 106% 166% 18 L70M D117N H134Q S212A M223E S331Y 124%  76%151% 29 L70M Y79S N108D D117N F124Y H134Q D202Q M223E L280I G309D 183% 57% 151% 7 L70M Y79S D117N H134Q M223E V261A L280I G309D E320T 141%124% 143% 21 L70M N108D D117N H134Q M223E G309W E320T 187% 151% 138% 5L70M D117N H134Q D156V L280I 135% 108% 137% 16 L70M H134Q D156V M223EL280I G309W 136% 131% 137% 3 L70M D117N H134Q M223E V256I L280I G309DS331Y K379P 109%  75% 135% 10 L70M D117N H134Q S212A M223E L280I G309WQ346E 101%  79% 133% 15 L70M K77T D117N H134Q S212A M223E V256I L280IG309D 154% 130% 129% 26 L70M Y79S D117N M223E L280I K379P 137% 123% 129%12 L70M V109L D117N H134Q L224V L280I G309D  98%  71% 128% 19 L70M V109LD117N F124Y H134Q M223E V261A L280I G309W  96%  98% 128% 27 D117N H134QM223E L224V V256I L280I 132% 102% 127% 13 L70M D117N H134Q D202Q M223EV261A L280I 116% 144% 126% 6 L70M K77T V90L D117N H134Q D202Q M223EL280I G309D 135% 113% 124% 11 L70M D117N H134Q D156V M223E L280I G309DE320T Q346E 153% 101% 119% 23 L70M D117N H134Q M223E G309D Q346E V367IK379P  67% 102% 118% 1 L70M Y79S D117N H134Q M223E V256I L280I G309DQ346E 132% 116% 117% 9 L70M D117N F124Y H134Q M223E L238I L280I G309DV367I 105%  97% 115% 25 L70M D117N H134Q L238I L280I G309W K379P  97% 92% 113% 14 L70M D117N D202Q M223E L224V L280I G309D  85% 126% 111% 4L70M D117N H134Q S212A M223E V261A L280I G309D V367I  83% 115% 108% 22V109L D117N H134Q M223E L238I L280I G309D E320T 110%  93% 106% 24 L70MN108D D117N V261A L280I G309D  95% 117% 102% Camel wt 100% 100% 100%

Statistical Analysis of the Positional and Mutational Effects on C/P

A statistical, PCA based, analysis was used to identify single mutationswith positive effects on the specificity of milk clotting over generalcasein proteolysis (C/P) of camel chymosin. The following mutations werefound to be beneficial for high C/P ratios:

S331Y, Y79S, K77T, D117N, H134Q, N108D, G309W, L224V, D156V, L280I,M223E, V367I, F114Y

Example 11: Evaluation of Camel Variants

A statistical, PCA based, analysis was performed on the combined set ofvariants from Examples 9 and 10, and single mutations were identifiedwith positive effects on the specificity of milk clotting over generalcasein proteolysis (C/P) of camel chymosin. The following mutations werefound to be beneficial for high C/P ratios:

F281A, H134Q, I103V, S331Y, S222G, I154L, L280I, G309D, D117N, L224V,N108D, L163E, G309W, K77T, Y79S

These mutations agree well with the beneficial mutations determined inExamples 9 and 10.

Structural Evaluation of Positional and Mutational Effects on C/P

As seen in Example 8, the majority of beneficial mutations are againlocated distant from the substrate binding cleft. Only L280I and F281Aare located directly in the cleft (Gilliland et al. 1990). 1280 pointsinto the hydrophobic core of the C-terminal lobe. This mutation mighttherefore lead to subtle conformational changes of the binding cleftand, thus, influence substrate specificity. Position 281 is part of theS2 binding site and interacts with the P2 position in the caseinsubstrate. A mutation in this position is very likely to have an impacton casein binding and, thus, proteolysis. Mutations G309W and S331Y arepositioned on the surface of the C-terminal lobe in a region that hasbeen described to interact with κ-casein to aid substrate binding in thecatalytic cleft (Gilliland et al. 1990). These mutations might thereforehave a positive impact on substrate binding. I154L and D156V, and L163Erepresent changes to the core of the N-terminal lobe, possibly leadingto subtle structural rearrangements of the enzyme with impact oncatalytic activity. Mutations S222G and L224V introduce changes into thebeta sheet that might interact with the protein N-terminus in itsactivated form (Langholm Jensen et al.). Potential effects on theactivation state of the enzyme could result in shifted casein substratespecificity. The remaining hit mutations K77T, Y79S, I103V, N108D,D117N, and H134Q are located on the surface of the N-terminal lobe and,with exception of I103V, represent exchanges of polar amino acids. Thesechanges on the surface of the enzyme most probably influenceinteractions with casein molecules leading to improved specificity infavor of κ-casein.

REFERENCES

-   1: WO02/36752A2 (Chr. Hansen)-   2: Suzuki et al: Site directed mutagenesis reveals functional    contribution of Thr218, Lys220 and Asp304 in chymosin, Protein    Engineering, vol. 4, January 1990, pages 69-71-   3: Suzuki et al: Alteration of catalytic properties of chymosin by    site-directed mutagenesis, Protein Engineering, vol. 2, May 1989,    pages 563-569-   4: van den Brink et al: Increased production of chymosin by    glycosylation, Journal of biotechnology, vol. 125, September 2006,    pages 304-310.-   5: Pitts et al: Expression and characterisation of chymosin pH    optima mutants produced in Tricoderma reesei, Journal of    biotechnology, vol. 28, March 1993, pages 69-83-   6: M. G. Williams et al: Mutagenesis, biochemical characterization    and X-ray structural analysis of point mutants of bovine chymosin,    Protein engineering design and selection, vol. 10, September 1997,    pages 991-997-   7: Strop et al: Engineering enzyme subsite specificity: preparation,    kinetic characterization, and x-ray analysis at 2.0 ANG resolution    of Val111phe site mutated calf chymosin, Biochemistry, vol. 29,    October 1990, pages 9863-9871-   8: Supannee et al: Site-specific mutations of calf chymosin B which    influence milk-clotting activity, Food Chemistry, vol. 62, June    1998, pages 133-139-   9: Zhang et al: Functional implications of disulfide bond,    Cys45-Cys50, in recombinant prochymosin, Biochimica et biophysica    acta, vol. 1343, December 1997, pages 278-286.-   10: WO2013/174840A1 (Chr. Hansen).-   11: WO2013/164479A2 (DSM).-   12: Langholm Jensen et al: Camel and bovine chymosin: the    relationship between their structures and cheese-making properties,    Acta Crystallographica Section D: Biological Crystallography, vol.    69, 2013, pages 901-913.-   13: Gilliland et al: The three-dimensional structure of bovine    chymosin at 2.3 Å resolution, Proteins, vol. 8, 1990, pages 82-101.

The invention claimed is:
 1. An isolated chymosin polypeptide variantobtained by a method comprising: producing and isolating a chymosinpolypeptide variant of a parent polypeptide having chymosin activity,wherein the amino acid sequence of the variant differs from the aminoacid sequence of the parent by having an alteration comprising asubstitution at at least one amino acid position of the parent sequencecorresponding to any of positions 77, 154, 212, 224, 246, and 367,wherein the corresponding position in the parent sequence is determinedby alignment of the parent sequence with SEQ ID NO:1 (bovine chymosin),thereby obtaining the isolated chymosin polypeptide variant, wherein thevariant has chymosin activity; wherein: the amino acid sequence of theparent polypeptide has at least 65% sequence identity with the aminoacid sequence from position 59 to position 381 of SEQ ID NO:1 (themature bovine chymosin polypeptide); and the variant has fewer than 30amino acid alterations in the region from amino acid position 59 toamino acid position 381 as compared to the mature polypeptide from aminoacid position 59 to amino acid position 381 of SEQ ID NO:1 or ascompared to the mature polypeptide from amino acid position 59 to aminoacid position 381 of SEQ ID NO:2, as determined by an alignment of theamino acid sequence of the variant with the amino acid sequence of SEQID NO:1 or SEQ ID NO:2, respectively; with the proviso that the variantis NOT a specific variant having any of the following sets ofsubstitutions based on the numbering of SEQ ID NO:1:Q246E+G309D+S329P+D337E; R125Q+G128N+H204R+Q246E+S284T;Y185F+R213Q+Q246E; D216S+L224V+V263I+F281V+G309D; Y79S+L224V+L311I; andR119S+L224V+T297S.
 2. An isolated chymosin polypeptide variant of aparent polypeptide having chymosin activity, wherein the amino acidsequence of the variant differs from the amino acid sequence of theparent by having an alteration comprising a substitution at at least oneamino acid position of the parent sequence corresponding to any ofpositions 77, 154, 212, 224, 246, and 367, wherein the correspondingposition in the parent sequence is determined by alignment of the parentsequence with SEQ ID NO:1 (bovine chymosin), wherein the variant haschymosin activity; and wherein: (i) the amino acid sequence of theparent polypeptide has at least 90% sequence identity with the aminoacid sequence from position 59 to position 381 of SEQ ID NO:1 (themature bovine chymosin polypeptide); and (ii) the amino acid sequence ofthe variant has less than 100% sequence identity with the amino acidsequence from position 59 to position 381 of SEQ ID NO:1; and (ii) thevariant has fewer than 30 amino acid alterations in the region fromamino acid position 59 to amino acid position 381 as compared to themature polypeptide from amino acid position 59 to amino acid position381 of SEQ ID NO:1, as determined by an alignment of the amino acidsequence of the variant with the amino acid sequence of SEQ ID NO:1;with the proviso that the variant is NOT a specific variant having anyof the following sets of substitutions based on the numbering of SEQ IDNO:1: Q246E+G309D+S329P+D337E; R125Q+G128N+H204R+Q246E+S284T;Y185F+R213Q+Q246E; D216S+L224V+V263I+F281V+G309D; Y79S+L224V+L311I; andR119S+L224V+T297S; wherein the variant has a chymosin activity giving ahigher clotting activity to proteolytic activity (C/P) ratio as comparedto the C/P ratio of bovine chymosin comprising the amino acid sequencefrom position 59 to position 381 of SEQ ID NO:1.
 3. The isolatedchymosin polypeptide variant of claim 2, wherein the amino acid sequenceof the parent polypeptide has at least 97% sequence identity with theamino acid sequence from position 59 to position 381 of SEQ ID NO:1; andthe amino acid sequence of the variant comprises fewer than 10 aminoacid substitutions as compared to the amino acid sequence from position59 to position 381 of SEQ ID NO:1.
 4. The isolated chymosin polypeptidevariant of claim 2, wherein the alteration comprises at least onesubstitution selected from K77T, I154L, S212A, L224V, Q246E, and V367I.5. The isolated chymosin polypeptide variant of claim 2, wherein thealteration comprises substitutions selected from:L280I+G309D+L224V+E320T+T235S; L280I+G309W+K77T+R324I;L280I+G309D+Q220S+L224V+H134Q; L280I+G309D+L224V+I103V+L238I;L280I+G309D+F124Y+Q346E+I154L; L280I+G309D+I154L+V261A+V367I;L280I+G309D+Y79S+T342S+I154L; L280I+G309D+Q346E+K77T+T235S;L280I+G309D+S331Y+L224V+Y326F; L280I+G309D+Y79S+L224V+S212A;L280I+G309W+S212A+V261A; L280I+G309D+V256I+V261A+K379P;L280I+G309D+S222G+R324V+I154L; L280I+G309D+V256I+V90L+E141S;L280I+G309W+K120Q+V367I; L280I+G309D+K120Q+Y326F+K77T;L280I+G309D+I154L+T235S+K379P; L280I+G309D+M223E+L224V+L273V; andD117N+H134Q+M223E+L224V+V256I+L280I.
 6. An isolated chymosin polypeptidevariant of a parent polypeptide having chymosin activity, wherein theamino acid sequence of the variant differs from the amino acid sequenceof the parent by having an alteration comprising a substitution at atleast one amino acid position of the parent sequence corresponding toany of positions 77, 154, 212, 224, 246, and 367, wherein thecorresponding position in the parent sequence is determined by alignmentof the parent sequence with SEQ ID NO:1 (bovine chymosin), wherein thevariant has chymosin activity; and wherein: (i) the amino acid sequenceof the parent polypeptide has at least 90% sequence identity with theamino acid sequence from position 59 to position 381 of SEQ ID NO:2; and(ii) the amino acid sequence of the variant has less than 100% sequenceidentity with the amino acid sequence from position 59 to position 381of SEQ ID NO:2; and (iii) the variant has fewer than 30 amino acidalterations in the region from amino acid position 59 to amino acidposition 381 as compared to the mature polypeptide from amino acidposition 59 to amino acid position 381 of SEQ ID NO:2, as determined byan alignment of the amino acid sequence of the variant with the aminoacid sequence of SEQ ID NO:2; with the proviso that the isolatedchymosin polypeptide variant is NOT a specific variant having any of thefollowing sets of substitutions based on the numbering of SEQ ID NO:1:Q246E+G309D+S329P+D337E; R125Q+G128N+H204R+Q246E+S284T;Y185F+R213Q+Q246E; D216S+L224V+V263I+F281V+G309D; Y79S+L224V+L311I; andR119S+L224V+T297S; wherein the variant has a chymosin activity giving ahigher clotting activity to proteolytic activity (C/P) ratio as comparedto the C/P ratio of camel chymosin comprising the amino acid sequencefrom position 59 to position 381 of SEQ ID NO:2.
 7. The isolatedchymosin polypeptide variant of claim 6, wherein the alterationcomprises substitutions selected from: L280I+G309D+L224V+E320T+T235S;L280I+G309W+K77T+R324I; L280I+G309D+Q220S+L224V+H134Q;L280I+G309D+L224V+I103V+L238I; L280I+G309D+F124Y+Q346E+I154L;L280I+G309D+I154L+V261A+V367I; L280I+G309D+Y79S+T342S+I154L;L280I+G309D+Q346E+K77T+T235S; L280I+G309D+S331Y+L224V+Y326F;L280I+G309D+Y79S+L224V+S212A; L280I+G309W+S212A+V261A;L280I+G309D+V256I+V261A+K379P; L280I+G309D+S222G+R324V+I154L;L280I+G309D+V256I+V90L+E141S; L280I+G309W+K120Q+V367I;L280I+G309D+K120Q+Y326F+K77T; L280I+G309D+I154L+T235S+K379P;L280I+G309D+M223E+L224V+L273V; and D117N+H134Q+M223E+L224V+V256I+L280I.8. A method for making a fermented milk product, a quark or a cheese,comprising adding an effective amount of the isolated chymosinpolypeptide variant according to claim 1 to milk, wherein the milk issoya milk, sheep milk, goat milk, buffalo milk, yak milk, lama milk,camel milk or cow milk.
 9. A method for making a fermented milk product,a quark or a cheese, comprising adding an effective amount of theisolated chymosin polypeptide variant according to claim 2 to milk,wherein the milk is soya milk, sheep milk, goat milk, buffalo milk, yakmilk, lama milk, camel milk or cow milk.
 10. A method for making afermented milk product, a quark or a cheese, comprising adding aneffective amount of the isolated chymosin polypeptide variant accordingto claim 6 to milk, wherein the milk is soya milk, sheep milk, goatmilk, buffalo milk, yak milk, lama milk, camel milk or cow milk.
 11. Theisolated chymosin polypeptide variant of claim 1, wherein thesubstitution comprises one or more selected from K77T, I154L, S212A,L224V, Q246E, and V367I.
 12. The isolated chymosin polypeptide variantof claim 11, wherein the substitution further comprises one or moreselected from Y79S; V90L; D102N; I103V; N108D; D117N; F114Y; K120Q;F124Y; H134Q; L163E; S222G; M223E; L238I; Q246E; V261A; K279V; L280I;F281A; R300D,E,S,T,N, and Q; R312D,E,S,T,N, and Q; E320T; R324V; D325Q;Y326F; K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y, and W; S331Y; Q346E;I361L; and K379P.
 13. The isolated chymosin polypeptide variant of claim1, wherein the alteration comprises substitutions selected from:D156V+Q246E+L280I; L280I+G309D+L224V+E320T+T235S;L280I+G309W+K77T+R324I; L280I+G309D+Q220S+L224V+H134Q;L280I+G309D+L224V+I103V+L238I; L280I+G309D+F124Y+Q346E+I154L;L280I+G309D+I154L+V261A+V367I; L280I+G309D+Y79S+T342S+I154L;L280I+G309D+Q346E+K77T+T235S; L280I+G309D+S331Y+L224V+Y326F;L280I+G309D+Y79S+L224V+S212A; L280I+G309W+S212A+V261A;L280I+G309D+V256I+V261A+K379P; L280I+G309D+S222G+R324V+I154L;L280I+G309D+V256I+V90L+E141S; L280I+G309W+K120Q+V367I;L280I+G309D+K120Q+Y326F+K77T; L280I+G309D+1154L+T235S+K379P;L280I+G309D+M223E+L224V+L273V; and D117N+H134Q+M223E+L224V+V256I+L280I.14. The isolated chymosin polypeptide variant of claim 4, wherein thesubstitution further comprises one or more selected from Y79S; V90L;D102N; I103V; K120Q; F124Y; H134Q; L163E; M223E; L238I; V256I; V261A;K279V; F281A; R300D,E,S,T,N,Q; R312D,E,S,T,N,Q; E320T; R324V; Y326F;K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; I361L; and K379P.15. The isolated chymosin polypeptide variant of claim 6, wherein thealteration comprises at least one substitution selected from K77T,I154L, S212A, L224V, Q246E, and V367I.
 16. The isolated chymosinpolypeptide variant of claim 15, wherein the substitution furthercomprises one or more selected from Y79S; V90L; D102N; I103V; K120Q;F124Y; H134Q; L163E; M223E; L238I; V256I; V261A; K279V; F281A;R300D,E,S,T,N,Q; R312D,E,S,T,N,Q; E320T; R324V; Y326F;K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; I361L; and K379P.17. The isolated chymosin polypeptide variant of claim 6, wherein theamino acid sequence of the parent polypeptide has at least 97% sequenceidentity with the amino acid sequence from position 59 to position 381of SEQ ID NO:2; and the amino acid sequence of the variant comprisesfewer than 10 amino acid substitutions as compared to the amino acidsequence from position 59 to position 381 of SEQ ID NO:2.
 18. Theisolated chymosin polypeptide variant of claim 6, wherein the alterationcomprises at least one substitution selected from K77T, I154L, S212A,L224V, Q246E, and V367I.